EMBO/sd-character-level-ner huggingface dataset:
Figure 4Schematic representation of the two Switch regions (I and II) within the Rab8a GTPase domain. Numbers denote the corresponding amino acids of human Rab8a (upper panel). Amino acid sequence alignments of the Switch I region of Rab family proteins (lower panel). Three conserved hydrophobic residues (Ile38, Ile41, and Ile43) in this region are indicated in red, while the other hydrophobic residues are indicated in orange. The three hydrophobic residues were substituted with serine and this construct was designated as the T22N-3IS mutant. Note that Ile38 and Ile43 of Rab8a are not conserved in Rab7. Kyte-Doolittle hydrophobicity plots of the complete amino acid sequence of human WT Rab8a and T22N-3IS mutant. The hydrophobicity peak within WT Switch I was abolished in the 3IS mutant (indicated within the red box). The numbers on the horizontal axis denote the corresponding amino acid positions in these proteins. Substitution of the hydrophobic residues of Switch I with hydrophilic residues (T22N 3IS) abolished its binding to BAG6 protein. Rab8a (T22N-3IS) mutant protein was highly stable, while Rab8a (T22N) protein was quite unstable in HeLa cells. Actin was used as a loading control. Rab8a (T22N-3IS) mutant was not subject to polyubiquitin co-precipitation, while Rab8a (T22N) protein was.
Figure 5Schematic diagram of the MIC19 protein. MIC19 contains an N-terminal myristoylation motif followed by a DUF37 domain (domain of unknown function) and a CHCH (coiled-coil helix-coiled-coil helix) domain, and is terminated by a diGly motif. Myristic acid was co-translationally attached to the N-terminal Gly2 residue by N-myristoyltransferases (NMTs) after removal of the initiating Met by methionine aminopeptidases. The N-terminal sequence of MIC19 is indicated at bottom with the myristoylation site, Gly2, and residues crucial for myristoylation labeled turquoise or underlined, respectively. Live-cell images of U2OS cells stably expressing GFP or GFP N-terminally tagged with the wild-type or mutant N-terminal motif of MIC19 (aa. 1-24). Scale bar = 20 µm. Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2, DNCul2 or IMP-1088 treatments. IMP-1088 inhibits NMTs. C and M denote cytosol and mitochondrial fractions, respectively. Tubulin and VDAC serve as fractionation quality controls. The relative abundance of cytosolic MIC19/tubulin was normalized to that of lane 1 and is indicated under each lane in red. Another replicate of this experiment is shown in Fig. EV3B. Abundance comparison of GFP N- or C-terminal tagged with the wild-type or mutant N-terminal (aa. 1-24) or C-terminal (a.a. 218-227) motif of MIC19 in cells with indicated treatments. Data from sh#1 is shown here. Live-cell images of U2OS cells expressing indicated GFP fusion proteins with or without DNCul2 and IMP-1088 treatments. The complete set of images containing degron mutant controls is shown in Appendix Fig. S3. Scale bar = 20 µm. Representative confocal immunofluorescence images of HA-tagged MIC19 variants with MitoTracker staining in U2OS cells. The fluorescence intensities along the dashed lines are shown as line profile graphs on the right. Scale bar = 20 µm.
OtofPga/Pga ribbon synapses exhibit shorter tethers in the MP-SV pool upon stimulation(A,B) Example virtual sections depict SVs with single membrane- (orange arrowhead) and presynaptic density (pink arrowhead) -attached tethers in resting wild-type.Figure 8Example virtual sections depict SVs with single membrane- (orange arrowhead) and presynaptic density (pink arrowhead) -attached tethers in resting wild-type. Magnification 12,000x; scale bar, 40 nm. 3D reconstructions of same tethered SVs (orange) as shown in (A) and (B); membrane-attached (A', yellow tether) and presynaptic density (PD) -attached (B', pink tether). Individual tether lengths are depicted. For clarity, ribbon (R) and AZ-membrane are shown in transparent. Scale bar, 20 nm. The graph depicts the mean length of single membrane-, presynaptic density-attached and multiple-tethers. Data are presented as mean ± SEM. Stimulated OtofPga/Pga synapses exhibit a shorter average tether length compared to stimulated wild-type. The cumulative probability distribution of MP-SVs plotted against the tether length. The red dotted lines represent the mean MP tether length in resting wild-type for each sub-pool. Total mean tether length in the MP-SV pool. Data are presented as mean ± SEM. Longer tethers are present upon stimulation in wild-type. Resting OtofPga/Pga ribbon synapses harbor the longest tethers, and tether where shorter at stimulated OtofPga/Pga synapses compared to wild-type. The red dotted lines represent the mean MP tether length in resting wild-type for each sub-pool.
Figure 4A. Schematic of the genetic system used to analyse inducible DSB repaired by SSA. Chr. V contains ura3-52 and URA3 (at the endogenous URA3 locus) separated by ~ 4 kb of DNA containing KAN (grey box, arrow above indicates the promoter) and an HO site (triangle). Galactose-inducible expression of the HO endonuclease leads to DSB formation at the HO site. After DSB repair via SSA, the majority of cells become Kans Ura- as the 766 bp homology between URA3 and ura3-52 (grey shadows) is predominantly used. B. Frequency of DSB repair via SSA in SRS2 and srs2Δ cells with and without functional HR in the assay based on the system shown in panel A. Average ± SD (n=4) is shown for each genotype. Strains used in B and D: NK4691-4693; NK4805-4808; NK5081-5084; NK5085-5091. C. Schematic of the quantitative SSA assay used in panel D. Grey shadow represents the annealing region of 766 bp present on both sides of a DSB. Numbers next to the one-ended arrows indicate distances (in bp) from the homology to the restriction sites, SalI and EcoRI, used to generate DNA fragment L analysed by Southern blotting. D. Fragment L formation in SRS2 and srs2Δ cells in the presence and absence of Rad51. Also see Figure EV3 for blot images. Average ± SD (n=4) is shown for each time point. E. A schematic for the basic DNA strand extension assay used in F-G. F. Rad51 inhibits DNA synthesis of phiX174 ssDNA substrate (0.5 nM) by Polδ. Increasing amount of Rad51 (0.03, 0.08, 0.15, 0.25, 0.5, 0.75, 1.5 µM) was incubated with the pre-loaded replication complex (RFC (17.5 nM), PCNA (10 nM) and DNA) and DNA synthesis was started by the addition of Polδ (10 nM) and nucleotides containing α-32P labelled dATP. G. Srs2(1-910) suppresses the inhibition of DNA synthesis by Rad51. The reaction was carried out the same as in F except of the increasing amount of Srs2(1-910) (5, 15, 50, 150 nM) was added to indicated reactions before start of DNA synthesis. The relative % of DNA synthesis is indicated.
Figure 7Schematic showing the two genetically modified mouse lines crossed to perform conditional deletion of Kitl in endothelial cells. Left: number of Ter119- Kit+ CD41+ CD16/32+ EMPs in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 YS, determined by flow cytometry. Data are the mean (±SD) of 8 control and 7 Tie2Cre::Kitl∆/∆ YS analyzed individually over 4 independent experiments. Tail somite range: 13-16. Middle: number of pre-HSC type I (Ter119- VE-Cadherin+ Kit+ CD41+ CD45-) and pre-HSC type II/HSC (Ter119- VE-Cadherin+ Kit+ CD41+ CD45+) in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 AGM+VU, determined by flow cytometry. Data are the mean (±SD) of 7 control and 5 Tie2Cre::Kitl∆/∆ AGM+VU analyzed individually over 3 independent experiments. Tail somite range: 14-15 (Kitlf/f, f/+); 14-16 (Tie2Cre::Kitl∆/∆). Right: number of Ter119- Kit+ CD41+ CD16/32+ EMPs and Ter119- Kit+ CD41- CD71+ CD44+ early erythroid cells in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 FL, determined by flow cytometry. Data are the mean (±SD) of 7 control and 5 Tie2Cre::Kitl∆/∆ FL analyzed individually over 3 independent experiments. Tail somite range: 13-16. CFU-C numbers in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 YS. Data are the mean (±SD) of 6 control and 3 Tie2Cre::Kitl∆/∆ YS plated in duplicate in 2 independent experiments. Tail somite range: 13-16. Analysis of long-term multi-lineage HSC potential in E11.5 Tie2Cre::Kitl∆/∆ and control Tie2Cre::Kitl∆/+ or Kitlf/f or f/+AGM+VU. Irradiated CD45.1 syngeneic mice were transplanted with 1 e.e. of AGM+VU cells. PB chimerism is represented as the percentage of donor CD45.2+ cells among total CD45+ cells, 16 weeks after transplant. A total of 29 recipients were transplanted with Tie2Cre::Kitl∆/+ or Kitlf/f or f/+ cells and 8 with Tie2Cre::Kitl∆/∆ cells, over 7 independent experiments. Kitlf/f or f/+ is represented with a circle, Tie2Cre::Kitl∆/+ a triangle and Tie2Cre::Kitl∆/∆ with a square. Tail somite range: 12-17 (control); 12-17 (Tie2Cre::Kitl∆/∆). CFU-C numbers in Tie2Cre::Kitl∆/∆ and control Kitlf/f or f/+ E11.5 Fl. Data are the mean (±SD) of 7 control and 5 Tie2Cre::Kitl∆/∆ FL plated in duplicate in 3 independent experiments. GEMM: granulocyte, erythroid, monocyte/ macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid. Tail somite range: 13-16. Number of phenotypic LSK, HPC and HSC per FL in E12.5 Tie2Cre::Kitl∆/∆and control (Kitlf/f or f/+) FL, determined by flow cytometry and total FL cell counts. Cells were gated as 7-AAD- Lin- (F4/80- CD3e- Nk1.1- Ter119- Gr-1- B220- CD19-). LSK: Lin- Sca1+ Kit+; HPC: Lin- Sca1+ Kit+ CD48+ CD150- and HSC: Lin- Sca1+ Kit+ CD48- CD150+. Gating strategy is shown in Figure EV4E. Data are the mean (±SD) of 12 control and 6 Tie2Cre::Kitl∆/∆ FLs analyzed individually over 3 independent experiments. Total cellularity of E12.5 Kitlf/f or f/+ (n=14) and Tie2Cre::Kitl∆/∆ (n=11) FL. Error bars represent SD.
a) Schematic for spatiotemporally controlled light-activated lysosomal damage.f1(b) AlPcS2a can be specifically targeted onto lysosomes. Using our staining procedure, AlPcS2a signals colocalized with Lysotracker Red (top panels) and lamp1-RFP (middle panels), but not with other cellular organelle markers such as EGFP-LC3B (bottom panels, marker for autophagic structures), in HeLa cells. Scale bars, 10 μm.
Fig.Figure 7Dynamin inhibitors (80 μM Dynasore or 20 μM Dyngo-4a) inhibit phagocytosis of opsonized 3-μm beads in WT BMDM; n = 101-149 cells, ***P<0.001. Rescue of the TPC-KO dynamin defect by the dynamin activator, Ryngo-1-23 (80 μM): mean number of internalised beads per cell as a percentage of the WT control; n = 128-241 cells, **P<0.01, ***P<0.001. In WT BMDM, expression of a dominant-negative dynamin-2 mutant (Dyn2 K44A) reduced phagocytosis (n = 68-184). Expression of wildtype dynamin-2 (Dyn2) or constitutively active dynamin-2 (S764A mutant, mimicking the dephosphorylated state) in Tpcn1-/- macrophages restored phagocytosis of 3-μm beads after 10 min, n = 10-76 cells; ***P<0.001 vs WT control, ### P<0.001 vs Tpcn1-/- control. Single WT BMDM expressing HA-tagged dynamin-2 (immunolabelled with anti-HA, in red) undergoing phagocytosis of IgG-3-μm beads (blue). Images were taken 10 or 30 min post-addition of beads; actin (green) labelled with Phalloidin Alexa 488. Cartoon indicating sites of action of pharmacological agents. Calcineurin inhibitors, FK506 (10 μM) or cyclosporin A (CsA, 10 μM) inhibited phagocytosis in WT cells, n = 65-159 cells. Ryngo (80 μM) rescued phagocytosis defects in Tpcn1-/- BMDM even in the presence of FK506 or CsA, n = 103-208 cells. Calcineurin activity and cytosolic Ca2+ responses simultaneously monitored in single RAW 247.6 using CaNAR2 and jRGECO1a, respectively, upon addition of 3-μm beads. CaNAR2 signals (FRET Y/C) were inhibited by FK506 (10 μM) and Ned-19 (10 μM); (H) single-cell traces and (I) mean maximum responses. n = 65-169; ***P<0.001 vs control. Phagocytosis induced dephosphorylation of dynamin-2 (ratio of phospho-dynamin to total dynamin-2 expressed as a percentage of WT at time zero) in WT BMDM, but not in Tpcn1-/- BMDM; **P<0.01, ***P<0.001 (time-point matched unpaired t-test) n = 10-15. Measured using in-cell western assay, representative wells of plate image: total dyn2 (red) and phospho-dyn2 (green). See also Appendix Figures S6 and S7.
AIN‐1 is selectively degraded by autophagy.Figure 3AIN‐1::GFP is weakly and diffusely expressed in the cytoplasm during embryogenesis in wild‐type animals. (A) Nomarski image of the embryo shown in (B). Scale bar, 10 μm for whole embryo. C. elegans embryos remain the same size during embryogenesis and loss of autophagy activity has no effect on the embryo size. Thus, scale bars for embryos are only shown once in each figure. (C) AIN‐1::GFP forms a large number of aggregates in epg‐6 mutant embryos. (A-C) are confocal images. (D) Western blotting shows that endogenous AIN‐1 protein levels are dramatically elevated in autophagy mutant embryos compared with wild type. (E) No AIN‐1::GFP aggregates are formed in sqst‐1 mutant embryos. (F) AIN‐1::GFP accumulates into numerous aggregates in epg‐7 mutant embryos. (G,H) Number of AIN‐1::GFP (G) and ALG‐2::GFP (H) aggregates per focal plane in the indicated strains. Error bars indicate the s.d. of four examined embryos. (I,J) ALG‐2::GFP is diffusely localized in the cytoplasm in wild‐type embryos. (I) Nomarski image of the embryo shown in (J). (K) ALG‐2::GFP forms a few aggregates in epg‐6 mutant embryos. (L) ALG‐2::GFP forms a large number of aggregates in epg‐6 mutant embryos under stress conditions. GFP, green fluorescent protein; s.d., standard deviation.
induces hepatic lipid accumulation (A) Western blots and quantification of respective protein levels in liver of HFD-fed mice treated with or without ASK1 inhibitor (n=4 mice per treatment).Figure 6Western blots and quantification of respective protein levels in liver of HFD-fed mice treated with or without ASK1 inhibitor (n=4 mice per treatment). Representative images of liver sections stained with H&E (scale bar represents 100 µm) liver triglyceride (TG) content of HFD-fed mice treated with or without ASK1 inhibitor (n=8 mice per treatment).
SID 26681509 inhibits the 1,25D3 mediated inhibition of HIV and M.Figure 7SID 26681509 inhibits the 1,25D3 mediated inhibition of HIV and M. tuberculosis replication.MDM were pretreated with SID 26681509 (SID) before treatment with 1,25D3 and subsequent infection with HIV and/or M. tuberculosis (TB). (A) ELISA performed for HIV p24 antigen release at days 0, 4 and 7. B) Intracellular mycobacteria were harvested and assayed for mycobacterial growth by cfu enumeration at day 0 and 7. (C) Intracellular mycobacteria harvested and assayed for viability using the MGIT 960 based on time to positivity at day 0 and 7. All bar and line graphs are reported as mean ± s.e.m. of three independent experiments performed in triplicate. § P<0.05; * P<0.01.
PI(5)P Is a Master Regulator of Autophagy in Response to Glucose Starvation (A) HeLa cells stably expressing GFP-LC3 were shifted to starvation media (HBSS or glucose-free DMEM) for 4 hr (in the presence of 200 nM Wm or 100 nM YM-201636).Figure 7(A) HeLa cells stably expressing GFP-LC3 were shifted to starvation media (HBSS or glucose-free DMEM) for 4 hr (in the presence of 200 nM Wm or 100 nM YM-201636).(B) Quantification of numbers of GFP-LC3 vesicles per cell in HeLa cells stably expressing GFP-LC3 treated as in (A) is shown in the graph (mean ± SEM). (C) Western blot analysis of LC3-II and tubulin levels in MEF and SKNSH cells were treated as in (A) in complete media or glucose-free media in the presence of 100 nM YM-201636, 200 nM Wm, and 400 nM BAF for 4 hr. (D) HeLa cells transfected with GFP-PHD3X and Strawberry-ATG16L1 for 16 hr were left in complete media (basal) or glucose-free media (glucose free) for 4 hr in the presence or absence of 100 nM YM-201636, then fixed and imaged on a confocal microscope. (E and F) HeLa cells transfected with GFP-PI5P4K2A, 2B, 2C, or PI5P4K2C catalytic dead and RFP-LC3 for 30 hr (E) were starved for glucose for 4 hr, then fixed and imaged on a confocal microscope. Bar, 10 μm. (F) Quantification of cells (percentage of total) showing more than ten autophagic vesicles (RFP-LC3 vesicles) in the different conditions from (F) is shown in the graph; n = 200 cells (mean ± SEM). (G) HeLa cells treated with CTR or MTMR3 siRNA for 5 days were transfected for the last 16 hr with GFP-PHD3X, Strb-ATG16L1, and myc-MTMR3 WT. Cells were starved for glucose for 4 hr, then fixed, stained with anti-myc antibodies, and imaged on a confocal microscope. Bar, 10 μm. (H) HeLa cells transfected for 16 hr with GFP-PHD3X and Strb-ATG16L1 were starved for 4 hr in HBSS or glucose-free media, then fixed, stained for PI(3) P antibodies, and imaged on a confocal microscope. Bar, 10 μm. See also Figure S7.
Figure 2Schematic showing replacement of wild-type SecA in a translocation intermediate by Walker B (WB) SecA mutants (green). WB SecA with additional mutations (WT, Y794G, R342E) was added to preformed intermediates (IM) and incubated for the indicated times before proteolysis. The gel is representative of two replicates.   Quantification of (B). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show average of replicates. Colors correspond to WB SecA carrying additional mutations, as shown in the inset box.
Figure 1A Regulation of svh-2 expression in response to axon injury. Axon injury initiates cAMP signaling and the Ca2+-p38 MAPK pathway, which together function to induce the formation of an ETS-4-CEBP-1 transcription factor complex. This complex binds to the svh-2 promoter to induce svh-2 expression. MXL-1 forms a complex with MDL-1. B Structure of MXL-1. Schematic diagrams of MXL-1 and its mammalian counterpart Max are shown. The domains (B-HLH-LZ) shown are the basic region (B; blue), helix-loop-helix (HLH; yellow) and leucine zipper (LZ; red). The bold line underneath indicates the extent of the deleted region in the tm1530 deletion mutant. C Representative D-type motor neurons in wild-type and mxl-1 mutant animals 24 h after laser surgery. In wild-type animals, a severed axon has regenerated a growth cone (arrow). In mutants, proximal ends of axons failed to regenerate (arrowhead). Scale bar = 10 μm. D Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% confidence intervals (CI). Data information statistical significance was determined by Fisher's exact test; *P < 0.05; ***P <0.001; NS; not significant.
Ub and p62 accumulate in α- and β′-COP–depleted 293/GFP-LC3 cells.Figure 5.Ub and p62 accumulate in α- and β′-COP-depleted 293/GFP-LC3 cells. (A) 293/GFP-LC3 cells were treated with control or β′-COP siRNA for 48 h, then fixed and labeled with anti-p62 and anti-ubiquitin antibodies for colocalization with GFP-LC3. (B) HEK293 cells treated as in A and labeled with anti-ubiquitin and anti-p62 antibodies. (C) Cryosections from β′-COP-depleted cells were labeled with anti-Ub antibodies followed by 10-nm protein A-gold. AV, autophagosomes.
Figure 3A. Sub-cellular localization of Nox4. Tunicamycin (Tn 2 µg/ml, 6h) increased Nox4 levels in H9c2 cells as assessed by spinning disk confocal microscopy (scale bar, 10 µm).3D SIM images (scale bars, 2 µm) showed localisation of Nox4 (green) to the ER, which was labeled with an anti-KDEL antibody (red). At higher magnification (right), yellow dots denote co-localization of Nox4 and KDEL signals. Cell nuclei were stained with DAPI (blue). 1 Z slice from 3D stack is shown. B. Progressive enrichment of GADD34, PP1, eIF2α and Nox4 in membrane fractions of tunicamycin-treated H9c2 cells. C. After sucrose gradient fractionation of lysates of tunicamycin-treated H9c2 cells, GADD34, PP1 and Nox4 co-eluted in fractions 12 and 13 (F12, F13). D. Immunoprecipitation (IP) of pooled fractions 12/13 with an anti-GADD34 antibody revealed the presence of both PP1 and Nox4. E. The association of Nox4 with GADD34 was validated in HEK293 cells co-transfected with Nox4 and either Flag-tagged or non-tagged GADD34, followed by IP with an anti-Flag antibody. F. Co-transfection of HEK293 cells with GADD34-Flag and different myc-tagged Nox4 constructs, followed by IP with an anti-myc antibody. GADD34 binds to full-length Nox4 (FL) and the Nox4 transmembrane domain (TD) but not the C-terminal domain (CD). G. Representative pseudocolor images of simultaneous ER and cytosolic ROS measurement with HyPer ER and HyPerRed Cyto, respectively, in tunicamycin-treated MEF cells. Redox-insensitive mutant probes were used as negative controls and to exclude pH changes. Extracellular H2O2 (200 nM) was added as a positive control. The pseudocolor scale is shown along the left vertical edge of each image. KO = Nox4-/-. Scale bars, 2 µm. H. Transfection of HEK293 cells with PP1 and GADD34 increased PP1 activity (bar graph) and decreased phospho-eIF2 levels (immunoblots). Co-transfection of full-length Nox4 reduced PP1 activity and increased phospho-eIF2 levels (captions at bottom refer both to the bar graphs and immunoblots). These effects were abrogated when either Nox4 P437H or the Nox4 transmembrane domain (TD) were transfected. Nox4 did not affect phosphorylation of glycogen synthase (GS-P) or histone H3 (H3-P). All experiments were n=3/group. Values below the immunoblots are mean ± SEM levels for phospho-eIF2/total-eIF2. *, p<0.05 comparing third and fourth lanes; Levels of significance for comparisons of PP1 activities are shown above the bar columns. See also Fig EV1 and Appendix Fig S4.
Figure 2HeLa cells were treated with DMSO (Vehicle) or 0.03 mg/ml digitonin (+Digitonin), fixed and then double immunostained for PGAM5 and Tom20. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PGAM5 and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student's HeLa cells stably expressing FLAG-Stx17 wild type (WT) were transfected with a plasmid encoding Su9-GFP (mitochondria) or Sec61β-GFP (ER). At 24 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PLA dots and Su9-GFP or Sec61β-GFP. Values are means ± SEM (n = 3). ***P<0.001 (paired Student's t-test). HeLa cells were treated with DMSO (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and subjected to Percoll-based fractionation. Equal amounts of proteins were analyzed by IB using the indicated antibodies. PNS, postnuclear supernatant; MS, microsomes; Mt, mitochondria. The amounts of proteins recovered on fractionation were as follows for vehicle and CCCP treatment, respectively: PNS (6.6 mg and 5.5 mg), cytosol (4.8 mg and 4.9 mg), MS (2.0 mg and 2.2 mg), MAM (0.55 mg and 0.46 mg) and Mt (0.30 mg and 0.28 mg). Electron microscopic analysis of HeLa cells expressing PGAM5-GFP and APEX2-GFP-binding peptide. Arrows indicate the position of 3,3'-diaminobenzidine reaction at the ER-mitochondria interface. Scale bar, 500 nm. HeLa cells stably expressing FLAG-Stx17 WT were mock-transfected or transfected with siRNA for Mfn1, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test). HeLa cells were mock-transfected or transfected with siRNA for Mfn1, Stx17, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against Drp1 and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).