EMBO/soda-nel huggingface dataset:
Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis (2015)
Figure 6

Figure 6 - 3D recordings of apoptotic mitochondria suggest the formation of a Bax-ring induced pore in the mitochondrial outer membrane.

Shown are seven optical sections through a mitochondrion of an apoptotic U2OS cell. Top and middle row: Tom22 and Bax (recorded in STED) channel, respectively. Bottom row: overlay (Tom22: red; Bax: green). Note the absence of Tom22 signal in the area enclosed by the Bax-ring. Displayed are raw data. Scale bar: 0.5 µm.
Gustatory receptor 28b is necessary for avoiding saponin in Drosophila melanogaster (2018)
Figure 2

Figure 2. Gr28b was required for saponin-mediated avoidance and ingestion.

A Binary food choice assay with 26 Gr mutants for 5% saponin (n ≥ 4).

B Concentration-dependent avoidance of saponin in control, Gr28bMi and Gr28b Df/ Gr28bMi (n ≥ 4).

C Amount of ingestion calculated at the indicated time points (15, 30, 60, and 240 min) using spectrophotogram analysis for control and 5% saponin-containing food (n = 8).

D, E PER assay for control and Gr28bMi flies in the (D) legs or (E) proboscis. Flies were initially given 2% sucrose, then 2% sucrose in combination with 5% saponin (n = 4).

The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis as a post hoc test to compare two sets of data (*P < 0.05, **P < 0.01).
Gustatory receptor 28b is necessary for avoiding saponin in Drosophila melanogaster (2018)
Figure 3

Figure 3. S-type and I-type sensilla in labellum mediated saponin avoidance.

A Mapping analysis of all the sensilla following stimulation with 5% saponin in control and Gr28bMi flies (we followed Tanimura's nomenclature) (n ≥ 8).

B Sample traces of saponin-induced action potentials on S6 sensilla.

C Tip recording analysis of S6 sensilla from control and Gr28bMi flies. Stimuli used are 0.2% DEET, 1.0 mM coumarin, 0.5 mM quinine, 10 mM umbelliferone, 10 mM caffeine, 0.3 mM strychnine, 0.3 mM lobeline, 0.1 mM chloroquine, 0.2 mM denatonium, 0.5 mM papaverine, 0.1 mM berberine and 0.1% SDS (n ≥ 7).

D Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene (hid), under Gr33a-GAL4 or Gr66a-GAL4. All heterozygote controls (UAS-hid/+, Gr33a-GAL4/+, and Gr66a-GAL4/+) are shown (n ≥ 4).

E Tip recording analysis of the indicated S-type sensilla and genotypes (n ≥ 7).

The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (*P < 0.05, **P < 0.01).
Gustatory receptor 28b is necessary for avoiding saponin in Drosophila melanogaster (2018)
Figure 4

Figure 4. The structure of Gr28b genome and specific requirement of GR28b(C) for saponin-mediated avoidance and electrophysiology.

A The genomic structure of Gr28b. It is composed of five different forms by alternative splicing. The arrow head indicates the P-element insertion. White boxes indicate predicted untranslated regions in flybase. Arrows indicate primers for RT-PCR.

B RT-PCR analysis with the indicated primers in A for control and Gr28bMi flies with tubulin as a control. From left to right, the band of tubulin of wild-type, tubulin of Gr28bMi, DNA ladder, Gr28b of wild-type, and Gr28b of Gr28bMi.

C, D Each isoform from Gr28b.a to Gr28b.e was driven by Gr28b.a-GAL4 or Gr28b.c-GAL4 in a Gr28bMi background. The asterisks indicate significant differences from that of the Gr28b mutant detected by a single-factor ANOVA with Scheffe's analysis (**P < 0.01).

C Binary food choice assay for recovery experiments with each of the five alternative Gr28b isoforms using the GAL4/UAS systems. (n ≥ 4).

D Saponin-induced action potential frequencies from the indicated transgenic flies on S6 sensilla (n = 7).
Gustatory receptor 28b is necessary for avoiding saponin in Drosophila melanogaster (2018)
Figure 5

Figure 5. The expression of Gr28b.c-GAL4 reporter in bitter-sensing GRNs.

A Binary food choice assays were conducted after GRNs were ablated by expressing the cell death gene (hid), under Gr28b.c-GAL4 (n = 4).

B Tip recording analysis of the indicated S-type sensilla and genotypes (n ≥ 7).

The error bars represent SEMs. The asterisks indicate significant differences from that of the control detected by a single-factor ANOVA with Scheffe's analysis to compare two sets of data (**P < 0.01).

C Expression of the Gr28b.c-GAL4 reporter in subsets of Gr66a-positive GRNs. First panel shows that all bitter responsive GRNs are labeled by Gr66a-I-GFP (anti-GFP, green). Second panel shows the expression of the Gr28b.c-GAL4, crossed with UAS-DsRed (anti-DsRed, red). Third panel shows merged image of first and second panels. Forth panel shows a schematic diagram of sensilla arrangement. Red-marked sensilla indicate the expression of Gr28b.c-GAL4 reporter in second panel. Scale bars represent 40 μm.
Gustatory receptor 28b is necessary for avoiding saponin in Drosophila melanogaster (2018)
Figure 6

Figure 6. Saponin toxicity during adult stage and larval growth.

A, B Survival rate of (A) control and (B) Gr28bMi adult raised on control and the indicated concentrations of saponin-containing cornmeal diet (n = 4). Statistics and LD50 are presented in Appendix Table S1.

C, D Survival rate based on binary food choice. control and Gr28bMi were allowed to feed 100 mM fructose versus 100 mM (C) or 200 mM fructose (D) mixed with 5% or 7.5% saponin. Statistics and LD50 are presented in Appendix Table S2. (n = 4)

E, F Cumulative pupation rate of (E) control and (F) Gr28bMi larvae raised on the indicated saponin-containing food as well as control food (n = 4). The error bars represent SEMs. The asterisks indicate significant differences from normal diet fed which were detected by a single-factor ANOVA with Scheffe's analysis (*P < 0.05, **P < 0.01).
Phosphorylation of mitophagy and pexophagy receptors coordinates their interaction with Atg8 and Atg11 (2013)
Figure 1

Atg30 interacts with Atg8 through a cryptic AIM, and phosphorylation upstream of the AIM regulates their interaction. (A) IP of GFP–Atg8 (α‐GFP), Atg30–Flag (α‐Flag) and Pex3 (α‐Pex3) under pexophagy conditions. The abundant peroxisome matrix protein, AOX, was used as a negative control. Input: total lysate; ϕ: IP without antibody. (B) Two multiple sequence alignments, including 11 Atg30 homologues and 11 Atg32 homologues (identical residues are indicated with black boxes, and similar residues with grey boxes), and a sequence logo of the combined multiple sequence alignments from Atg30 and Atg32 homologues listed currently in GenBank. (C,D) Pexophagy experiments of Δatg30 (ϕ), wild‐type (Atg30) and Atg30 AIM mutant (Atg30W73A F76A) cells were done by fluorescence microscopy, following the degradation of peroxisomes labelled with BFP fused at its C‐terminus to the Ser–Lys–Leu peroxisomal targeting signal 1 (BFP–SKL) and biochemically by monitoring peroxisomal thiolase degradation. Vacuoles were labelled with FM4‐64. Scale bar, 5 μm. (E) AH109 cells were transformed with two yeast two‐hybrid assay plasmids, AD and BD, which encode the indicated domains fused with Atg30, Atg32 and Atg8 or an empty vector, as negative controls and grown on +His and −His+40 mM 3‐AT plates. (F). Δatg30 (ϕ) and Δatg30 cells complemented with Atg30–HA (Atg30) and several Atg30–HA mutants were immunoprecipitated (α‐HA IP) under pexophagy conditions. In addition, α‐HA IP of Δatg30 cells (ϕ) and Δatg30 cells complemented with Atg30–HA (Atg30) were incubated with (+) and without (−) λPP. Input: total lysate. (G) Pexophagy in atg30 mutants was monitored by following thiolase levels of oleate‐induced peroxisomes after shifting cells to SD‐N. aa, amino acid; AD, activation domain; AIM, Atg8‐family‐interacting motif; AOX, alcohol oxidase; BD, binding domain; GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; λPP, λ protein phosphatase.
Phosphorylation of mitophagy and pexophagy receptors coordinates their interaction with Atg8 and Atg11 (2013)
Figure 2

Interactions between Atg30, Atg8 and Atg11. (A1) Atg30 sequences from aa 68–122 of wild‐type (Atg30), the S71A mutant (Atg30S71A) and two different deletions of the sequence between the AIM and phosphosite required for Atg11 binding in Atg30 (Atg30WDILSSS and Atg30WSILSSS). (A2) Pexophagy experiments of Δatg30 cells complemented with appropriate wild‐type or mutant Atg30 proteins described in A1. (A3) Two‐hybrid assays between Atg30 wild‐type or mutants (described in A1) and Atg8. Phosphomimic S71E was included to detect the interaction with Atg8. (B1) Schematic of the two Atg30 molecules, one with an Atg8‐binding site mutated and a second with an Atg11‐binding site (A11‐BS) mutated, used to complement Δatg30 cells by co‐expression. P: indicates phosphorylation in vivo. (B2) Pexophagy experiments of Δatg30 cells complemented with the two Atg30 molecules described in B1. (C1) Schematic of the Atg30 mutations, S71A and S112A that impair Atg8 and Atg11 binding, respectively. (C2) Pexophagy assays of Δatg30 cells complemented with Atg30 wild‐type and mutants. aa, amino acids; AD, activation domain; AIM, Atg8‐family‐interacting motif; BD, binding domain; HA, haemagglutinin.
Phosphorylation of mitophagy and pexophagy receptors coordinates their interaction with Atg8 and Atg11 (2013)
Figure 3

Atg8 and Atg11 localization during pexophagy. (A) Large phagophore membrane formation in WT and the Atg30 mutant cells monitored by GFP–Atg8 during pexophagy conditions. (B) Localization of GFP–Atg11 during pexophagy of methanol‐induced peroxisomes in cells expressing WT or mutant Atg30 proteins. White arrows indicate correct localization and yellow arrows indicate mislocalization, or in case of Atg8 localization, indicate absence of phagophore membrane elongation. Peroxisomes were labelled with BFP–SKL and vacuoles with FM4‐64. Scale bar, 5 μm. GFP, green fluorescent protein; WT, wild type.
Phosphorylation of mitophagy and pexophagy receptors coordinates their interaction with Atg8 and Atg11 (2013)
Figure 4

Atg32, ScAtg32 and Atg36 use similar interaction mechanisms as Atg30. (A) Mitophagy experiments of WT (PPY12), Δatg5, Δatg32 (ϕ) and Δatg32 complemented with WT Atg32 (Atg32), Atg32 with a deletion of the sequence between the AIM and phosphosite required for Atg11 binding (Atg32WQVLSSS), Atg32 AIM mutant (Atg32W121A V124A), mutants of the Thr upstream of the Atg32 AIM (Atg32T119A) and mutants altered in the Ser required for Atg11 binding (Atg32S159A). The cells were grown in YPL medium and shifted to SD‐N. Mitophagy was followed by the transport of Tom20–mCherry to the vacuole by fluorescence microscopy. Mitophagy was classified as −no mitophagy,+few cells show mitophagy, +++ most cells show mitophagy and ++++ almost all the cells show mitophagy (the intensity and numbers of cells containing Tom20–mCherry in the vacuole was considered). Scale bar, 5 μm. (B) P. pastoris Δatg32 cells (ΔPpatg32) expressing Tom20–GFP and expressing the indicated Atg32 mutants were cultured in YPL medium for 12, 18 and 36 h. Mitophagy was monitored by GFP appearance by immunoblotting with α‐GFP antibodies. (C) N‐terminal sequence of Atg36 manually aligned against the several sequence alignments of Atg30, Atg32 and ScAtg32. Identical residues are indicated with black boxes, and similar residues with grey boxes. (D) Two‐hybrid protein–protein interaction analysis of ScAtg36, ScAtg8 and ScAtg11. The receptors were mutated at the AIM (Atg36F33A L36A), at serine(s) upstream of the AIM (Atg36S31A) and at the Atg11‐binding site (Atg36S97A). (E) S. cerevisiae Δatg36 cells (ΔScatg36) expressing thiolase–GFP and expressing the indicated Atg36 mutants were cultured in oleate medium until mid‐log growth and then shifted to SD‐N. Pexophagy was monitored by GFP appearance by immunoblotting with α‐GFP antibodies. (F) Y3H analysis of ScAtg36 with ScAtg8 and ScAtg11. The Y3H technology is on the basis of the yeast two‐hybrid system but with the co‐expression of third protein as a competitor and indicated in the figure (NLS–ScAtg11 or NLS–ScAtg8). The positive control was the ScAtg36 mutant affected in Atg11 binding (ScAtg36S97A) or in Atg8 binding (ScAtg36S31A), which should be unaffected by the competition of NLS–ScAtg11 or NLS–ScAtg8, respectively. Appropriate auto‐activation and interaction controls were also included. AD, activation domain; AIM, Atg8‐family‐interacting motif; BD, binding domain; GFP, green fluorescent protein; WT, wild type; Y3H, yeast three‐hybrid.
Ubiquilin4 is an adaptor protein that recruits Ubiquilin1 to the autophagy machinery (2013)
Figure 1

Ubqln1 interacts with Ubqln4. (A) Inducible GFP‐S‐Ubqln1 293 cell line was treated with 1 μg/ml of doxycycline for 24 h. GFP‐S‐Ubqln1 was immunoprecipitated and sent for mass spectrometry analysis. Red sequences indicate Ubqln4 peptides identified by mass spectrometry. (B) 293 cells were co‐transfected with GFP‐S or GFP‐S‐Ubqln1 and HA‐Ubqln4. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies. Lower molecular weight GFP‐tag‐containing bands that appear in the GFP‐S‐Ubqln1 lane might represent degradation products. (C) Endogenous Ubqln1 in 293 cells was immunoprecipitated using a rabbit Ubqln1 antibody or a control rabbit IgG. The immunoprecipitates were analysed by western blotting using anti‐Ubqln1 and anti‐Ubqln4 antibodies. (D) Confocal images of Hela cells transfected with GFP‐S‐Ubqln1 and RFP‐Ubqln4. Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross‐section. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; Ubqln, Ubiquilin; WCL, whole‐cell lysate.
Ubiquilin4 is an adaptor protein that recruits Ubiquilin1 to the autophagy machinery (2013)
Figure 2

Ubqln4 interacts with LC3. Confocal images of (A) Hela cells transfected with GFP‐LC3 and RFP‐Ubqln4 and starved for 1 h or (B) Hela cells starved for 1 h and stained for endogenous LC3 and Ubqln4. The squares show colocalization. The insets show the enlargement of the indicated areas. Linescan of the white line in the merged image indicates the intensities of the fluorescent molecules in the cross‐section. (C) 293 cells were transfected with GFP‐S, GFP‐S‐Ubqln1 or GFP‐S‐Ubqln4 and treated with 50 μM chloroquine for 14 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies. (D) Recombinant His‐Ubqln1, His‐Ubqln4, His‐S5a or His‐p62 was mixed with LC3 covalently coupled to agarose beads. The beads were washed, eluted and detected by western blot using anti‐His antibody. The extent of Ubqln1, Ubqln4, s5a or p62 co‐immunoprecipitation with LC3 was measured. Bars for Ubqln1, Ubqln4 and s5a represents the average and s.e.m. of four independent experiments. *P‐value=0.0094. (E) 293 cells were transfected with GFP‐S‐Ubqln4 WT, GFP‐S‐Ubqln4 ΔSTI1 12, GFP‐S‐Ubqln4 Δ between STI1 or GFP‐S‐Ubqln4 ΔSTI1 34 and treated with 50 μM chloroquine for 14 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies. GFP, green fluorescent protein; IP, immunoprecipitation; LC3, microtubule‐associated protein light chain 3; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.
Ubiquilin4 is an adaptor protein that recruits Ubiquilin1 to the autophagy machinery (2013)
Figure 3

Ubqln1 punctate distribution is dependent on Ubqln4. (A) Confocal images of Hela cells transfected with GFP‐S‐Ubqln1 and also transfected with either control or Ubqln4 siRNA. The cells were either untreated or were transferred to starvation medium for 1 h. (B) Quantification of the number of GFP‐S‐Ubqln1 WT puncta per cell in (A). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. *P‐value=0.011, **P‐value=0.0005 and ***P‐value=0.0001. (C) Depletion of Ubqln4 with siRNA in (A) was verified by western blot using anti‐Ubqln1, anti‐Ubqln4 and anti‐tubulin antibodies. (D) Hela cells transfected with GFP‐S‐Ubqln1 and also transfected with either control or Ubqln4 siRNA were starved for 1 h. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐LC3 antibodies. (E) Confocal images of GFP‐S‐Ubqln1 WT in Hela cells transfected with RFP, RFP‐Ubqln4 or RFP‐Ubqln4 ΔUBL. (F) Quantification of the number of GFP‐S‐Ubqln1 WT puncta per cell in (E). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. *P‐value=0.0001 and **P‐value=0.0003. GFP, green fluorescent protein; IP, immunoprecipitation; LC3, microtubule‐associated protein light chain 3; UBL, ubiquitin‐like; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.
Ubiquilin4 is an adaptor protein that recruits Ubiquilin1 to the autophagy machinery (2013)
Figure 4

Ubqln1 interacts with Ubqln4 through its UBA domain. (A) 293 cells were co‐transfected with GFP‐S‐Ubqln1, GFP‐S‐Ubqln1 ΔUBL (deletion of UBL domain) or GFP‐S‐Ubqln1 ΔUBA (deletion of UBA domain) and HA‐Ubqln4. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies. (B) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln1, Ubqln1ΔUBL and Ubqln1ΔUBA. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average and s.e.m. of four independent experiments. *P‐value=0.0041. (C) 293 cells were co‐transfected with GFP‐S or GFP‐S‐Ubqln1 and HA‐Ubqln4, HA‐Ubqln4 ΔUBL or HA‐Ubqln4 ΔUBA. GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies. (D) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln4, Ubqln4ΔUBL and Ubqln4ΔUBA. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average and s.e.m. of four independent experiments. *P‐value=0.0003 and **P‐value=0.0001. (E) 293 cells were co‐transfected with GFP‐S‐Ubqln1 and HA‐Ubqln4, HA‐Ubqln4 ΔUBA or HA‐Ubqln4 ΔUBA I55A (I55 in the UBL domain of Ubqln4 was mutated to A). GFP was immunoprecipitated and analysed by western blot using anti‐GFP and anti‐HA antibodies. (F) The extent of Ubqln4 co‐immunoprecipitation with Ubqln1 was measured and the ratio of Ubqln4 to Ubqln1 was calculated for WT Ubqln4, Ubqln4ΔUBA and Ubqln4ΔUBA I55A. The numbers represent the ratio of Ubqln4 to Ubqln1 relative to WT. Each bar represents the average of two independent experiments. GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; UBA, ubiquitin‐associated; UBL, ubiquitin‐like; Ubqln, Ubiquilin; WCL, whole‐cell lysate; WT, wild‐type.
Ubiquilin4 is an adaptor protein that recruits Ubiquilin1 to the autophagy machinery (2013)
Figure 5

Depletion of Ubqln4 inhibits autophagosome–lysosome fusion. (A) Confocal images of Hela cells transfected with mCherry‐GFP‐LC3 and also transfected with control, Ubqln1 or Ubqln4 siRNA. The cells were either untreated or were transferred to starvation medium for 8 h. (B) Quantification of the number of mCherry/GFP double‐positive and mCherry single‐positive puncta per cell in (A). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. The number of mCherry single‐positive puncta of Ubqln1 and Ubqln4 siRNA was compared with that of control siRNA. *P‐value=0.0475 and **P‐value=0.0373. GFP, green fluorescent protein; LC3, microtubule‐associated protein light chain 3; Ubqln, Ubiquilin.
Rab12 regulates mTORC1 activity and autophagy through controlling the degradation of amino‐acid transporter PAT4 (2013)
Figure 1

Rab12 regulates autophagy. (A) Quantification of the amount of LC3‐II protein in mouse Rab‐knockdown cells. Representative blots are shown in supplementary Fig S1C online. Error bars represent the means and s.e.m. of three independent experiments. (B) Quantification of the amount of p62 protein in candidates Rab‐knockdown cells. Representative blots are shown in supplementary Fig S1D online. Error bars represent the means and s.e.m. of three independent experiments. (C,D) Control and Rab12‐knockdown MEFs were cultured under N or S conditions, fixed and then immunostained with the antibodies indicated. Representative images are shown in supplementary Fig S2 online. The mean numbers of LC3‐positive (C) or Atg16L1‐positive (D) dots per cell are shown. Error bars represent the means and s.e.m. of representative data (n≥80) from three independent experiments. (E) MEFs that had been transfected with the control siRNA or Rab12 siRNA were cultured as in (C,D), and their lysates were analysed by immunoblotting with the antibodies indicated. (F) Quantification of (E). (G) Control and Rab12‐knockdown MEFs were cultured under N or S conditions in the absence or presence of 100 μM bafilomycin A1 for 1 h. Cell lysates were analysed by immunoblotting with the antibodies indicated, and the intensity of the LC3‐II band was quantified. The normalized amount (arbitrary units) of LC3‐II in lanes 1, 2, 3, 4, 5 and 6 is 1.0, 2.3, 4.3, 0.9, 1.6 and 3.8, respectively. (H) MEFs transiently expressing mStr‐Rab12 were cultured under starved conditions and immunostained with the antibodies indicated. Scale bar, 20 μm. MEFs, mouse embryonic fibroblasts; N, nutrient‐rich; S, starved; siRNA, short interfering RNA. *P0.05; **P0.01; ***P0.005.
Rab12 regulates mTORC1 activity and autophagy through controlling the degradation of amino‐acid transporter PAT4 (2013)
Figure 2

Effect of Rab12 knockdown on mTORC1 activity. (A) Control and Rab12‐knockdown MEFs were cultured under nutrient‐rich or starved conditions, fixed and then immunostained with the antibodies indicated. Scale bar, 20 μm. (B) Control and Rab12‐knockdown MEFs were cultured as in (A), and their lysates were analysed by immunoblotting with the antibodies indicated. (C) Lysates of MEFs that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with the antibodies indicated. (D,E) Quantification of the phospho‐S6K levels and phospho‐Akt levels shown in (C). MEFs, mouse embryonic fibroblasts; N, nutrient‐rich; S, starved; siRNA, short interfering RNA. *P0.05; **P0.01.
Rab12 regulates mTORC1 activity and autophagy through controlling the degradation of amino‐acid transporter PAT4 (2013)
Figure 3

Rab12 regulates constitutive degradation of amino‐acid transporter PAT4. (A) MEFs transiently co‐expressing Myc‐PAT4 and mStr‐Rab12 were immunostained with anti‐Myc antibody. (B) MEFs transiently expressing Myc‐PAT4 were immunostained with the antibodies indicated. Scale bars, 20 μm. (C) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA‐PAT4 were analysed by immunoblotting with the antibodies indicated. (D) Lysates of MEFs stably expressing HA‐PAT4 that had been transfected with control or Rab12 siRNAs were analysed by immunoblotting with the antibodies indicated. (E) Quantification of (D). ***P0.005. (F) PAT4 mRNA levels from MEFs transfected with control or Rab12 siRNA as revealed by reverse‐transcription PCR analyses. Gapdh was used as an internal control. (G) Total cell lysates and surface biotinylated proteins from MEFs stably expressing HA‐PAT4 that had been transfected with control or Rab12 siRNA were analysed by immunoblotting with the antibodies indicated. Note that the amounts of plasma‐membrane‐localized PAT4 protein and TfR protein were higher in Rab12‐knockdown MEFs, whereas the total amounts of EGFR protein and plasma membrane‐localized EGFR protein in these cells were slightly lower, but the mechanisms responsible for these changes are unknown. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; MEFs, mouse embryonic fibroblasts; PAT4, proton‐coupled amino‐acid transporter 4; siRNA, short interfering RNA.