EMBO/soda-nel huggingface dataset:
LONP1 Targets HMGCS2 to Protect Mitochondrial Function and Attenuate Chronic Kidney Disease (2022)
Figure 6

Figure 6. Effect of HMGCS2 on mitochondrial function and renal fibrosis.

A Immunohistochemical analysis of HMGCS2 expression in CKD children. Scale bar: 50μm.

B Immunohistochemical semi-quantitative IOD analysis of HMGCS2 (n=8 in Control group, n=15 in CKD group).

C Pearson correlation analysis of HMGCS2 and atrophy and fibrosis score (AFS) in renal biopsy specimens.

D Western blot analysis for the expression of HMGCS2 in UUO model.

E Densitometric analysis for the expression of HMGCS2 in UUO model (n=3, biological replicates).

F, G Western blot and densitometric analysis of HMGCS2 in WT and cKI mice of UUO model (n=3 or 4, biological replicates).

H, I qRT-PCR and western blotting analysis of Hmgcs2 expression in WT and Hmgcs2+/- mice (n=3, biological replicates).

J Fibrotic area statistics of Sirius red staining in WT and Hmgcs2+/- mice of UUO model (n=12 in WT groups, n=8 in Hmgcs2+/- groups, biological replicates).

K Deposition of total fibrosis in kidney tissues in WT and Hmgcs2+/- mice after UUO was determined by Masson's trichrome staining. Scale bar: 50μm.

L Sirius red staining of WT and Hmgcs2+/- mice of UUO model. Scale bar: 50μm.

M, N Western blot and densitometric analysis for the expression of FN1, α-SMA and Collagen III in WT and Hmgcs2+/- mice after UUO (n=6 in control groups, n=8 in UUO groups, biological replicates).

O qRT-PCR analysis of FN1, α-SMA and Collagen III in WT and Hmgcs2+/- mice after UUO (n= 10 or 12 in WT groups, n=8 in Hmgcs2+/- groups, biological replicates).

P Succinate dehydrogenase (SDH) staining in WT and Hmgcs2+/- mice after UUO. Scale bar: 100μm.

Data information: In (B, E, H, I), data are presented as mean ± SEM. Student's t-test. In (G, J, N, O), data are presented as mean ± SEM. One-way ANOVA.
LONP1 Targets HMGCS2 to Protect Mitochondrial Function and Attenuate Chronic Kidney Disease (2022)
Figure 7

Figure 7. The anti-fibrotic response effect of a novel LONP1 activator in mPTC cells.

A 10 high docking score molecules showed LONP1 activating effect in cell free LONP1 protease activity assay system, of which 84-B10 showed stronger activity. Bortezomib (Bor), a reported LONP1 inhibitor, is involved as a contrast.

B 84-B10 dose dependently induced the peptidase activity of LONP1 as estimated by detecting the fluorogenic di-peptide substrate (Z-AA)2-Rh110 (n=6 or 3, biological replicates).

C Chemical structure of 84-B10.

D Proposed binding model of 84-B10 binding to the human LONP1 catalytic domain. Three and two-dimensional illustrations of the interaction between 84-B10 and human LONP1 catalytic domain.

E Physical interaction between 84-B10 and LONP1 performed by Surface Plasmon Resonance (SPR).

F Protein levels of HMGCS2 and LONP1 were estimated in mPTCs treated with different concentration of 84-B10 or vehicle (0.1% DMSO) for 24 h.

G, H Representative immunoblot and densitometric analysis of HMGCS2, FN1, Collagen III, α-SMA and Vimentin in mPTCs treated as indicated. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n=3 in each group).

I qRT-PCR analysis of Lonp1, Collagen I, Collagen III, α-SMA, Vimentin and Periostin transcript levels in mPTCs treated as indicated (n=4 in each group, biological replicates).

J-N Representative Western blot and densitometric analysis of LONP1, FN1, α-SMA and Vimentin of each group was shown. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n=3 in each group). mPTCs were transfected with siNC or siLONP1, and then pre-treated with 84-B10 (10 μM) 2h before TGF-β1 (10ng/mL) treatment for another 24h.

Data information: Data are presented as mean ± SEM. Student's t-test.
Species‐specific impact of the autophagy machinery on Chikungunya virus infection (2013)
Figure 1

Autophagy components promote CHIKV infection and control virus‐induced cell death in HeLa cells. Cells were mock infected or infected and immunoblotted for actin, p62 (A) or LC3 (B). Cells were infected for 15 h and labeled using antibodies to p62 and capsid (C). Cells were transfected with GFP‐LC3‐B, infected for 15 h and labeled with anti‐capsid antibody (D). Mice were infected for 3 days, then whole‐cell lysates of muscle were immunoblotted for LC3 or actin (E) and muscle sections were labeled with p62 and capsid antibodies (F). Cells were treated with DMSO (CTRL) or rapamycin and then infected. Viral replication (G) and production (H) were assessed and whole‐cell lysates were immunoblotted for capsid or actin (I). Cells were treated with CTRL, Beclin1 (left) or Atg7 (right) siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (K) and production (L) were assessed and whole‐cell lysates of siRNA‐treated cells were immunoblotted for Beclin1, Atg7, capsid and actin (M). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. CHIKV, Chikungunya virus; CTRL, control; GFP, green fluorescent protein; DMSO, dimethylsulphoxide; siRNA, short interfering RNA.
Species‐specific impact of the autophagy machinery on Chikungunya virus infection (2013)
Figure 2

Autophagy receptors p62 and NDP52 localize to distinct pools of capsid and have distinct effects on CHIKV infection in Hela cells. Cells were infected for 15 h and labeled using antibodies to NDP52 and capsid (A), to p62, NDP52 and capsid (B), to NDP52, LC3‐C or capsid (C), to p62, ubiquitin (FK2) and capsid (D), or to NDP52, ubiquitin (FK2), and capsid (E). Cells were treated with CTRL or p62 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (F), viral replication (left) and qRT‐PCR (right) (G), and viral production (H) were assessed and whole‐cell lysates were immunoblotted for p62, capsid or actin (I). Cells were treated with CTRL or NDP52 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (left) and qRT‐PCR (right) (K) and production were assessed (L). Whole‐cell lysates of siRNA‐treated cells were immunoblotted for NDP52, capsid or actin (M). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. C, capsid; CHIKV, Chikungunya virus; CTRL, control; NDP52, nuclear dot protein 52 kDa; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; siRNA, short interfering RNA.
Species‐specific impact of the autophagy machinery on Chikungunya virus infection (2013)
Figure 3

p62 associates with ubiquitinated capsid and controls cell death induced by cytotoxic capsid in HeLa cells. Cells were infected for 24 h and immunoprecipitation experiments were performed using antibodies to FK2, capsid or nonspecific IgG controls. Immunoprecipitated proteins were revealed using antibodies to FK2 or capsid (A,B). Cells were transfected with Ub‐HA and capsid‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. using antibodies to FLAG or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐HA antibody (C). Immunoprecipitation experiments were performed using antibodies to p62 or nonspecific IgG controls. Immunoprecipitated proteins were revealed using antibodies to p62, FK2 or capsid (D,E). Cells were infected for 3 h and then transfected with empty plasmid (CTRL) or p62WT‐3XFLAG or p62deltaUBA‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. with anti‐FLAG antibody. Immunoprecipitated proteins were revealed using antibodies to FLAG or capsid (F). Cells were treated with CTRL or p62 siRNA, then infected for 24 h. Immunoprecipitation experiments were performed using antibodies to FK2 or nonspecific IgG controls. Immunoprecipitated proteins were revealed using anti‐capsid antibody (G). Cells were infected for 15 h and labeled with antibodies to p62, LAMP‐1 or capsid (H). Cells were infected for 9, 15 or 24 h and treated with DMSO (CTRL) or bafilomycin 3 h before the end of experiment. Whole‐cell lysates were immunoblotted for capsid or actin (I). HeLa cells were transfected with empty plasmid (CTRL), control protein (cherry‐3XFLAG) or capsid‐3XFLAG at fixed (750 ng) or at the indicated concentrations. Cell mortality (that is, % of dead cells) was measured 24 h post transfection (J,K). Cells were treated with CTRL or p62 siRNA and transfected with NT or capsid‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 48 h post transfection (L). Cells were infected with CHIKV for 3 h and transfected with empty plasmid (CTRL), p62WT‐3XFLAG, p62deltaUBA‐3XFLAG or p62deltaLIR‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 24 h post transfection (M). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. CHIKV, Chikungunya virus; CTRL, control; IP, immunoprecipitation; NT, empty plasmid; p.i., post infection; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; siRNA, short interfering RNA; Ub, ubiquitin; WB, western blot.
Species‐specific impact of the autophagy machinery on Chikungunya virus infection (2013)
Figure 4

Human NDP52 binds nsP2, localizes near CHIKV RCs, and restricts cell shutoff promoting cell survival in HeLa cells. Yeast cells expressing Gal4 DNA BD fused alone (empty vector) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to NDP52 or the indicated NDP52 deletion mutants (top). Yeast cells expressing BD fused to nsP2 or the indicated nsP2 deletion mutants were cotransformed with a plasmid encoding the Gal4 AD fused alone (empty vector) or to NDP52 (bottom) (A). Cells were infected for 15 h, then labeled using antibodies to NDP52 and nsP2 (B), or to TGN46, nsP2 and nsP3 (C). Cells were treated with control (CTRL) (left) or NDP52 (right) siRNA, infected for 15 h, then labeled using antibodies to NDP52, nsP2 and puromycin. Quantitative analysis was performed by counting the percentage of infected cells with TGN‐associated RCs (n=30 cells per experiment) (D). Cells were transfected with empty plasmid (CTRL) or nsP2wt‐3XFLAG or nsP2R606A‐3XFLAG. Cell mortality (that is, % of dead cells) was measured 24 h post transfection (E). Cells were treated with control (CTRL) or NDP52 siRNA, then mock infected (M) or infected (C) for 24 h. Cytoplasmic and nuclear fractions were isolated and immunoblotted for nsP2 and NDP52. The amounts of the cytoplasmic and nuclear fractions of nsP2 were quantified by densitometry and expressed as the ratio of nuclear to cytoplasmic fraction of nsP2 (F). Cells were treated with control (CTRL) or NDP52 siRNA, infected for 15 and 24 h and treated with puromycin. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for NDP52, capsid or actin. Puromycin incorporation into newly synthesized protein was revealed using antibodies to puromycin (G). Cells were treated with CTRL or NDP52 siRNA, then transfected with NT or nsP2‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 48 h post transfection (H). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. AD, transactivation domain; BD, binding domain; CHIKV, Chikungunya virus; CTRL, control; NDP52, nuclear dot protein 52 kDa; nsP, nonstructural protein; NT, empty plasmid; RC, replicative complexes; siRNA, short interfering RNA; TGN, trans‐Golgi network.
Species‐specific impact of the autophagy machinery on Chikungunya virus infection (2013)
Figure 5

NDP52 promotes CHIKV infection in a species‐specific manner. MEF cells were treated with CTRL or NDP52 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (A) and viral production (B) were measured. Yeast cells expressing Gal4 DNA BD fused alone (empty vector) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to hNDP52 or mouse NDP52 (C). MEF cells were infected for 3 h and transfected with empty plasmid (CTRL) or hNDP52‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. using antibodies to FLAG or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐nsP2 antibody (D). MEF cells were infected for 3 h and transfected with empty plasmid (CTRL), hNDP52, hLC3‐C or hNDP52 and hLC3‐C. Viral production (E) and cell mortality (F) were assessed 24 h post transfection. Isogenic wt or Atg5−/− MEFs were infected for 24 h and cell mortality (that is, fold change relative to mock‐infected cells) (G) and viral production (H) were assessed. MEF cells were infected and immunoprecipitation experiments were performed 24 h p.i. using antibodies to p62 or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐capsid antibody (I). MEF cells were treated with CTRL or p62 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J) and viral production were assessed. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for p62 or actin (K). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. AD, transactivation domain; BD, binding domain; CHIKV, Chikungunya virus; CTRL, control; hLC3‐C, human LC3‐C; hNDP52, human NDP52; IP, immunoprecipitation; MEF, mouse embryonic fibroblast; mNDP52, mouse NDP52; NDP52, nuclear dot protein 52 kDa; nsP, nonstructural protein; p.i., post infection; siRNA, short interfering RNA; WB, western blot.
Drosophila Rae1 controls the abundance of the ubiquitin ligase Highwire in post-mitotic neurons (2011)
figf1

(a) The Hiw-associated complex was purified by TAP (see Online Methods) and analyzed by one-dimensional SDS-PAGE gel followed by Sypro Ruby staining. Mass spectrometry identified the full-length Hiw, β-tubulin, Rae1 and Fsn, indicated by arrows. (b) The Rae1-associated complex purified from fly brains by TAP was analyzed by one-dimensional SDS-PAGE gel followed by Coomassie blue G-250 staining. Mass spectrometry identified Hiw, Nup98 and Rae1, indicated by arrows. (c) Larval brain lysate of wild-type (WT), hiw null mutant (hiwΔN) and Fsn mutant (Fsnf06595/Df(2R)7872) flies was subject to co-immunoprecipitation with antibody to Hiw (Hiw2b). Both the input and the immunoprecipitated complexes were analyzed by western blots with antibodies to Hiw or Rae1. The membrane was also blotted with antibody to β-tubulin to ensure equal amount of total proteins in the input samples. Full-length blots are presented in Supplementary Figure 8.
Drosophila Rae1 controls the abundance of the ubiquitin ligase Highwire in post-mitotic neurons (2011)
figf2

(a) The gene structure of Rae1. The black-arrowed boxes indicate exons. A transposable element, P{GawB}NP3499, located ∼400 bp upstream of the first exon of Rae1 was used to generate imprecise excision mutants of Rae1. PCR analysis indicates that ∼900 bp and a ∼1.5-k bp DNA fragment in the Rae1 promoter region are missing in Rae1EX28 and Rae1EXB12, respectively. (b) Western blot analysis on Rae1 protein in the brain lysate of wild-type, Df(2R)5764/+, Rae1EX28/+, or late second instar Rae1EX28, Rae1EX28/Df(2R)5764(/Df) and Rae1EXB12 /Df(2R)5764 larvae. β-tubulin was used as the loading control. Full-length blots are presented in Supplementary Figure 9. (c) Quantification of Rae1 protein levels in the Rae1 heterozygous and homozygous mutant brains. Rae1 protein levels were first normalized to β-tubulin protein levels and are presented as percentage of wild-type level. *P 0.05, **P 0.001. Error bars denote s.e.m.
Drosophila Rae1 controls the abundance of the ubiquitin ligase Highwire in post-mitotic neurons (2011)
figf3

(a) Representative confocal images of segment A3 muscle 6/7 synapses stained for both DVGLUT (green) and FasII (red), in late 2nd/early 3rd instar wild-type, Rae1EX28, Rae1EX28 presynaptic rescue (Rae1EX28; elav-Gal4/UAS–NTAP-Rae1) and Rae1EX28 wallenda suppression (Rae1EX28; wnd3) larvae. Scale bar represents 10 μm. (b,c) Quantification of bouton number (b) and size (c) in wild-type, Rae1EX28/Df, Rae1EX28, Rae1EX28 rescue, Rae1EX28; wnd3 suppression and hiwΔN larval NMJs (segment A3 muscle 6/7; n = 20, 21, 18, 20, 20 and 11, respectively, for bouton number; n = 271, 465, 548, 726, 486 and 826, respectively, for bouton size). There was no significant difference in either bouton number or bouton size between Rae1EX28 and Rae1EX28/Df (P > 0.1 for both comparisons). *P 0.001. Error bars denote s.e.m.
Drosophila Rae1 controls the abundance of the ubiquitin ligase Highwire in post-mitotic neurons (2011)
figf4

(a) Representative confocal images of muscle 4 synapses stained for both DVGLUT (green) and FasII (red) in wild-type, Rae1EX28/+, hiwND51, hiwND51; Rae1EX28/+, hiwΔN and hiwΔN; Rae1EX28/+ larvae. Scale bar represents 10 μm. (b) Quantification of bouton number at muscle 4 NMJs in wild-type, Rae1EX28/+, hiwND51, hiwND51; Rae1EX28/+, hiwΔN and hiwΔN; Rae1EX28/+ larvae (n = 27, 23, 42, 46, 23 and 25 cells, respectively). There was a significant increase in the number of boutons formed in hiwND51; Rae1EX28/+ compared with hiwND51 3rd instar larval NMJs (*P 0.001). The number of boutons in the hiwΔN; Rae1EX28/+ double mutant was not significantly different from that in hiwΔN (**P > 0.5), demonstrating a lack of enhancement of the hiwΔN phenotype by Rae1EX28/+. Error bars denote s.e.m.
Drosophila Rae1 controls the abundance of the ubiquitin ligase Highwire in post-mitotic neurons (2011)
figf5

(a) Schematic presentation of NTAP-tagged (NT-) and HM-tagged (HM-) hiw transgenes encoding full-length, mutated or truncated Hiw proteins. Hiw functional domains are marked with colored boxes. The positions of amino-acid substitution in NT-HiwΔRING and the added amino acid residue necessary to maintain the reading frame in NT-Hiw-RCC1 and NT-Hiw-PHR are indicated. The right column summarizes the ability of the given hiw transgene to rescue the synaptic overgrowth phenotype in hiw mutants, to cause dominant negative overgrowth phenotype when expressed in wild-type background and to bind to Rae1. (b) Western blot analysis of fly brain lysate using antibody to TAP (PAP, Sigma) or Myc revealed the expression of wild-type and mutant hiw transgenes in predicted size. (c) Representative confocal images of muscle 4 synapses stained for DVGLUT (green) and FasII (red) in wild-type (+/hiwND8; +/+; GS-elav-Gal4/+), NT-Hiw overexpression (+/hiwND8; UAS-NT-hiw/+; GS-elav-Gal4/+), NT-Hiw-NT overexpression (+/hiwND8; UAS-NT-hiw-NT/+; GS-elav-Gal4/+), NT-Hiw-CT1000 overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-CT1000), NT-Hiw-PHR overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-PHR), HM-Hiw-HindIII overexpression (+/hiwND8; UAS-HW-hiw-HindIII/+; GS-elav-Gal4/+), NT-Hiw-RCC1 overexpression (NT-hiw-RCC1/hiwND8; +/+; GS-elav-Gal4/+), or NT-Hiw-CT overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-CT) 3rd larval NMJs. Overexpression is indicated by an up arrow. Scale bar represents 10 μm. (d) Quantification of the number of boutons at muscle 4 NMJs in wild-type larvae or larvae overexpressing wild-type or mutant hiw transgenes (n = 23, 24, 26, 27, 26, 21, 25 and 27, respectively; *P 0.001). Error bars denote s.e.m.
Drosophila Rae1 controls the abundance of the ubiquitin ligase Highwire in post-mitotic neurons (2011)
figf6

(a) Indicated NTAP-tagged hiw transgenes (described in Fig. 5) and a TAP only transgene were expressed in neurons under the control of the BG380-Gal4 driver. Larval brain lysates from each sample were subject to IgG pulldown. Both the inputs and the IgG pulldown complexes were analyzed by western blot using antibody to Rae1. Rae1 was present in all of the pulldown complexes except for those from TAP only, NT-Hiw-CT1000 and NT-Hiw-NT. Full-length blots are presented in Supplementary Figure 10. (b) Quantification of the interaction between various Hiw transgenic proteins and Rae1. Rae1 intensities in TAP pulldown blots were normalized to both the intensities and molecular weights of the corresponding TAP pulldown proteins. (c) Representative confocal images of muscle 4 synapses stained for both DVGLUT (green) and FasII (red) in wild-type (BG380/Y;+/+; +/+), NT-Hiw-CT and GAP-GFP (BG380/Y; UAS-GAP-GFP/+; UAS-NT-hiw-CT/+), and NT-Hiw-CT and Rae1 (BG380/Y; +/+; UAS-NT-hiw-CT/UAS-GFP-Rae1) larvae. Scale bar represents 10 μm. (d) Quantification of number of boutons at muscle 4 NMJs in wild-type, NT-Hiw-CT overexpression (BG380/Y; +/+; UAS-NT-hiw-CT/+), NT-Hiw-CT and GAP-GFP, and NT-Hiw-CT and Rae1 (n = 24, 27, 32 and 26, respectively) larvae. Error bars denote s.e.m.; *P 0.001.
Drosophila Rae1 controls the abundance of the ubiquitin ligase Highwire in post-mitotic neurons (2011)
figf7

(a) Neuronal expression of neither Hiw nor Rae1 rescues the synaptic terminal overgrowth phenotype caused by the loss of function of the other gene. Quantification of the number of boutons in wild-type, Rae1EX28, Rae1EX28; Hiw rescue (Res) (Rae1EX28; elav-Gal4/UAS-GFP-hiw), hiwΔN and hiwΔN; Rae1 rescue (hiwΔN; elav-Gal4/UAS-NTAP-Rae1) larval NMJs (segment A3 muscle 6/7; n = 12, 12, 12, 7 and 11, respectively). (b) Representative confocal images of wild-type (BG380-Gal4/+; +/+; UAS-GFP-hiw/+) or Rae1 mutant (BG380-Gal4/+; Rae1EX28; UAS-GFP-hiw/+) ventral ganglions with UAS-GFP-hiw trangene expressed in neurons. The larvae were stained with antibodies to horseradish peroxidase (HRP, red) and GFP (green). Scale bar represents 10 μm. (c) Western blots of total proteins extracted from wild-type, hiwΔN, Rae1EX28 and Rae1EX28 rescue (Rae1EX28; elav-Gal4/UAS-NTAP-Rae1) larval brains probed with indicated antibodies. Full-length blots are presented in Supplementary Figure 11. (d) Western blot analysis on total proteins extracted from hiwΔN, wild-type (C155-Gal4/+) and Rae1 expression (C155-Gal4/+; UAS-NTAP-Rae1/+) larval brains. Full-length blots are presented in Supplementary Figure 12. (e) Quantification of Hiw protein levels in the wild-type and the Rae1 expression larval brains (*P 0.001, based on seven independent experiments). Arrows indicate a 38-kDa endogenous Rae1 protein and the arrowheads indicate the 58-kDa transgenic NTAP-Rae1 protein. Error bars denote s.e.m.
Drosophila Rae1 controls the abundance of the ubiquitin ligase Highwire in post-mitotic neurons (2011)
figf8

(a) Representative confocal images of segment A3 muscle 6/7 synapses stained for both DVGLUT (green) and FasII (red), in wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ wandering larvae. Scale bar represents 10 μm. (b) Quantification of the number of boutons in wild-type, atg1/+, atg2/+, atg18/+, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval NMJs (segment A3 muscle 6/7; n = 12, 12, 12, 12, 12, 16, 12 and 21, respectively). (c) Western blot analysis on total proteins extracted from wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval brains. The neural form of β-catenin (82 kDa, see Online Methods) was used as an internal control of neuronal proteins. Full-length blots are presented in Supplementary Figure 13. (d) Quantification of Hiw protein levels in wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval brains (based on six independent experiments). (e) Representative confocal images of muscle 4 synapses stained for both DVGLUT (green) and FasII (red) in wandering larvae of wild-type (C155-Gal4/+), Atg1 Gap-GFP (C155-Gal4/+; UAS-Gap-GFP/+; UAS-Atg16B/+), and Atg1 NTAP-Rae1 (C155-Gal4/+; UAS-NTAP-Rae1/+; UAS-Atg1/+). Scale bar represents 10 μm. (f) Quantification of the number of boutons at muscle 4 synapses in wild-type, Rae1 expression (C155-Gal4/+; UAS-NTAP-Rae1/+), Atg1 Gap-GFP and Atg1 NTAP-Rae1 wandering larvae (segment A2–4, n = 28, 27, 33 and 34, respectively). Error bars denote s.e.m. *P 0.001, **P 0.05.