EMBO/soda-nel huggingface dataset:
Parkin controls brown adipose tissue plasticity in response to adaptive thermogenesis (2019)
Figure 1

Figure 1. Parkin expression is repressed by thermogenic stimuli in brown and beige adipose tissue.

(A)Profile of transcript levels of autophagy-related genes in iBAT which are significantly modified in cold exposed mice (4ºC, 24h), compared with mice maintained at thermoneutral temperature (29ºC). (Fisher's exact test corrected by the Benjamini-Hochberg method, p< 0.05). Heatmap represents log-fold change of RPKM values of cold versus thermoneutral (TN) sample data obtained from RNAseq analysis. Each square represents replicates (n=4). The most repressed genes are shown on the bottom of the heatmap.

(B) Parkin expression in iBAT from mice exposed to cold (4ºC) for 1 day (1d) or 21 days (21d) compared with control mice (ctl) maintained at 22ºC. Left: Relative transcript levels. Right: representative immunoblot and Parkin protein quantification. Ponceau staining (PS) was used as loading control. (n=6; transcript levels) (n=3; protein levels). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, cold vs. control and ##p<0.01, 1d vs. 21d. One-way ANOVA with Tukey's post hoc test.

(C) Relative transcript levels of Park2 in sWAT and eWAT. (n= 6). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, cold vs. control and ###p<0.001, 1d vs. 21d. One-way ANOVA with Tukey's post hoc test.

(D) Relative transcript levels of Parkin in iBAT, sWAT and eWAT of mice injected with the β3-AR agonist CL316,243, every day for 1 week. (n=6). Data are presented as means ±s.e.m. *p<0.05, ***p<0.001, CL316,243 treatment vs. saline. Two-tailed unpaired Student's t-test.
Parkin controls brown adipose tissue plasticity in response to adaptive thermogenesis (2019)
Figure 2

Figure 2. Parkin expression is repressed by norepinephrine-induced, cAMP-mediated, thermogenic activation.

(A) Relative transcript levels of Park2 and Ucp1 in mature brown adipocytes compared with the stromal vascular fraction (SVF) in iBAT. (n=3). Data are presented as means ±s.e.m. **p<0.01, ***p<0.001. Two-tailed unpaired Student's t-test.

(B) mRNA expression profiles of Park2 and Ucp1 during differentiation. (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 compared to day (d) 4. Two-tailed unpaired Student's t-test.

(C) Relative transcript levels of Park2 and Ucp1 in brown adipocytes in culture with fetal bovine serum (FBS, to trigger in vitro differentiation) or charcoal-stripped serum (CSS, non-permissive for differentiation). (n=3). Data are presented as means ±s.e.m. **p<0.01, ***p<0.001. Two-tailed unpaired Student's t-test.

(D) Relative transcript levels of Park2 and Ucp1 in brown adipocytes in culture treated with norepinephrine (NE) or cAMP for indicated times. (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 compared to non-treated cells. One-way ANOVA with Dunnett's post hoc test.

(E) Parkin protein levels in brown adipocytes treated with NE or cAMP for the indicated times in the presence or absence of 3-methyladenine (3-MA). Top: Representative immunoblots. p62 and LC3BII proteins are shown as positive controls of 3-MA treatment. β-actin was use as loading control. Bottom: Parkin protein quantification (performed using 3 immunoblots processed in parallel due to sample size). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 compared to NE/cAMP non-treated cells. One-way ANOVA with Dunnett's post hoc test.

(F) Relative transcript levels of Park2 in brown adipocytes treated with NE in the presence or absence of cycloheximide (CHX). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001. Two-tailed unpaired Student's t-test.

(G) Relative mRNA expression levels of Parkin in brown adipocytes treated with NE in the presence or absence of actinomycin D (n=3). Data are presented as means ±s.e.m. One-way ANOVA with Tukey's post hoc test comparing treatments.

(H) Park2 gene promoter activity in HIB-1B cells transfected with a pGL4-Park2-Luc reporter construct treated with or without NE or cAMP. Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that in untreated cells. (n=6). Data are presented as means ±s.e.m. *p<0.05 compared to non-treated cells. ANOVA with Dunnett's post hoc test

(I) Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated with cAMP (12 h) plus H89 (a PKA inhibitor) or SB202190 (a p38 MAPK inhibitor). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 with vs. without inhibitor and #p<0.05, and ##p<0.01, ###p<0.001 with vs. without cAMP. Two-tailed unpaired Student's t-test.
Parkin controls brown adipose tissue plasticity in response to adaptive thermogenesis (2019)
Figure 3

Figure 3. Thermogenic activation represses Parkin through mechanisms involving lipolysis and PPARα pathway.

(A) Relative transcript levels of Park2, and concentration of glycerol in the culture medium of brown adipocytes treated with cAMP (24h) plus Atglistatin (an adipose triglyceride lipase inhibitor) or CAY10499 (a hormone sensitive lipase inhibitor). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 ***p<0.001 with vs. without inhibitor and #p<0.05, and ##p<0.01, ###p<0.001 with vs. without cAMP. Two-tailed unpaired Student's t-test.

(B) Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated for 24h with a PPARα activator (GW7647), a PPARα inhibitor (GW6471) with or without norepinephrine (NE), or all-trans-retinoic acid (RA). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 ***p<0.001. Two-tailed unpaired Student's t-test.

(C) Relative transcript levels of Park2 in wild-type and PPARα-KO brown adipocytes treated for 24h with NE (n=3). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.

(D) Park2 gene promoter activity in HIB-1B cells transfected with a pGL4-Park2-Luc reporter construct and, when indicated, with a pSG5-Ppara expression vector. Cells were treated with the PPARα activator GW7647 or NE. Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that only transfected with the pGL4-Park2-Luc construct (n=6). Data are presented as means ± s.e.m. *p<0.05 compared to non-treated cells. ANOVA with Dunnett's post hoc test.
Parkin controls brown adipose tissue plasticity in response to adaptive thermogenesis (2019)
Figure 4

Figure 4. Parkin-KO mice show over-activation of BAT and are protected against HFD-induced weight gain and insulin resistance.

(A) Left: Representative optical microscopic images of H&E-stained iBAT (scale bars, 110µm) and electron microscopic images of iBAT (scale bars, 20µm) from wild-type (WT) and Parkin-KO (KO) mice. Right: Quantification of lipid droplet area from electron microscopic images (10 images per condition were used, n =3 WT, n=3 Parkin-KO). Data are presented as means ±s.e.m. *p<0.05. Two-tailed unpaired Student's t-test.

(B) Representative thermal images (left) and quantification of surface temperature (right) for the interscapular region. (n=6). Data are presented as means ±s.e.m. ***p<0.001. Two-tailed unpaired Student's t-test.

(C) Representative immunoblot of UCP1 protein and the loading control (Ponceau staining, PS) in iBAT samples.

(D) Relative transcript levels of thermogenesis-related genes of iBAT from WT and Parkin-KO mice exposed to acute cold (4ºC, 24h) compared to those maintained at the control temperature (22ºC). (n=8; WT) (n=6; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001, CTL vs. cold; #p<0.05, WT vs. Parkin-KO. ANOVA with Tukey's post hoc test.

(E-G) Weight gain, blood glucose levels and plasma insulin levels, and insulin tolerance test (ITT) from WT and Parkin-KO mice fed with chow (CD) or high fat diet (HFD). (n=8; WT) (n=6; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001, CTL vs. cold; #p<0.05, WT vs. Parkin-KO. E and G were analyzed using the two-tailed unpaired Student's t-test. F was analyzed using ANOVA with Tukey's post hoc test.

(H) Relative transcript levels of thermogenesis-related genes in iBAT from WT and Parkin-KO mice given HFD. (n=8; WT) (n=6; Parkin-KO). Data are presented as means ±s.e.m. #p<0.05. Two-tailed unpaired Student's t-test.

(I) Relative transcript levels of Park2 in iBAT from WT mice fed CD or HFD. (n=8; WT) (n=6; Parkin-KO). Data are presented as means ±s.e.m. Two-tailed unpaired Student's t-test.
Parkin controls brown adipose tissue plasticity in response to adaptive thermogenesis (2019)
Figure 5

Figure 5. Deacclimation to cold induces Parkin expression and autophagic degradation of mitochondria. WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d).

(A) Top: Total protein content in iBAT during cold deaclimation. (n=6). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 compared to 21d. ANOVA with Dunnett's post hoc test. Bottom: Representative optical microscopic images of H&E-stained iBAT (scale bars, 100µm) and electron microscopic images of iBAT (scale bars, 10µm).

(B) Relative transcript levels of thermogenesis-related genes and Park2 in iBAT. (n=6). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 compared to 21d. ANOVA with Dunnett's post hoc test.

(C) Representative immunoblot of the indicated thermogenic and autophagic proteins in iBAT during cold acclimation and deacclimation. PS: Ponceau staining.

(D) Representative immunoblot of Parkin phosphoSer65 and total Parkin in whole lysates of iBAT and after Parkin immunoprecipitation. Arrowheads indicate Parkin 52kDa band and asterisk indicates a non-specific band.

(E) Representative electron microscopic images of autophagic and mitophagic events in iBAT after 1 day of cold deacclimation. Yellow arrowheads indicate mitochondrial cristae, blue arrowheads indicate double-membrane autophagosomes, and red arrowheads indicate structures compatible with mitochondria-derived vesicles. LD: lipid droplet. Scale bars, 1 µm.
Parkin controls brown adipose tissue plasticity in response to adaptive thermogenesis (2019)
Figure 6

Figure 6. Responsiveness of Parkin-KO mice to cold-deacclimation. Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d).

(A) Representative immunoblot of LC3B in iBAT, and the loading control (Ponceau staining, PS).

(B) Immunoblot for p62 and OPTN (left) and its quantification (right) in iBAT. PS was used as the loading control. Arrowhead indicates p62 band and asterisk indicates a non-specific band. (n=4; cold) (n=3; deacclimation). Data are presented as means ±s.e.m. **p<0.01. ANOVA with Tukey's post hoc test.

(C) Quantification of the total protein amounts of the indicated OXPHOS protein subunits and UCP1 in iBAT. (n=4; cold) (n=3; deacclimation). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.

(D) Citrate synthase activity in iBAT protein extracts. (n=6; WT) (n=8; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.

(E) Mitochondrial DNA (mtDNA) content in iBAT relative to nuclear DNA (nDNA). (n=6; WT) (n=8; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.

(F) Representative electron microscopic images of mitochondrial integrity and autophagic/mitophagic events in iBAT after 1 day of cold-deacclimation. Yellow arrowheads indicate mitochondria cristae and blue arrowheads indicate double-membrane autophagosomes. Left panels scale bars, 2 µm. Right panels scale bars, 1 µm.

EXPANDED VIEW FIGURE LEGENDS
Structural basis for loading and inhibition of a bacterial T6SS phospholipase effector by the VgrG spike (2020)
Figure 1

Figure 1. The N-terminal extension of Tle1 is required for VgrG-mediated transport into target cells.

A. Cross-link connectivity map of SVgrG-Tle1H complex. The two proteins are represented as rectangles in which the residues number and known domains are indicated. Straight lines represent inter-molecular cross-links, while dotted lines represent intra-molecular cross-links. Only the cross-links identified in two independent experiments were considered. Raw and cured data are provided in Dataset EV1.

B. Schematic representation of an alignment of Tle1 orthologs using ClustalW and draw alignment tool in KEGG(Kanehisa et al, 2017). Red box, bit score ≥ 200; pink box, bit score ≥ 80-200. Ordered locus names of the identified genes are indicated on the right. Domains are represented in grey boxes (Hcp, PF05638; PAAR (P), PF05488; VgrG, PF05954, COG3501; DUF2345, PF10106). The N-terminal segment found in EAEC Tle1 protein and absent in the others is represented by a blue box.

C. Antibacterial competition assay. E. coli K12 recipient cells (W3110 gfp+, kanR) were mixed with the indicated EAEC attacker cells (1:4 ratio): WT, ∆T6SS-1 and ∆tle1-tli1 carrying pBAD18 and pBAD33 vectors, or producing the indicated proteins, and spotted on SIM 0.02 % arabinose agar plates for 4-h at 37°C. The image of a representative bacterial spot is shown. The number of recovered prey cells is indicated in the upper graph (in log of colony-forming units (cfu)). The black, dark grey and light grey circles indicate values obtained from three different spots, and the average is indicated by the bar. Western blot analysis of the production of Tle1V and Tle1∆1-26 V is shown in the inset. The experiment was performed in triplicate and a representative result is shown.

D. Co-immunoprecipitation assay. Lysates from E. coli K-12 W3110 cells producing VSVG-tagged Tle1 (Tle1V) or VSVG-tagged Tle1∆1-26 truncated variant (Tle1∆1-26 V) were mixed with lysates from W3110 cells producing FLAG-tagged VgrG (VgrGFL) or not (-, empty vector) and subjected to immunoprecipitation on anti-FLAG-coupled beads. The mixed soluble lysates (Input) and the immunoprecipitated material (IP) were subjected to 12.5% SDS-PAGE and immunodetected with anti-FLAG (upper panel) and anti-VSVG (lower panel) monoclonal antibodies. Molecular weight markers (in kDa) are indicated on the left. This experiment was performed in triplicate and a representative result is shown.
Structural basis for loading and inhibition of a bacterial T6SS phospholipase effector by the VgrG spike (2020)
Figure 5

Figure 5. Inhibition of Tle1 by VgrG.

A. Specific phospholipase A1 (PLA1) activity measurements of Tle1H, SVgrG-Tle1H complex, and isolated SVgrG. Thermomyces lanuginosus lipase (TLL) was used as positive standard for PLA1 activity. Mean values and standard deviation from at least three independent assays are shown. Statistical analysis relative to the Tle1 activity is indicated. ***P<0.001 (P=0,0001998), unpaired two-sample Fisher-Pitman permutation test.

B. VgrG inhibition of Tle1H. Specific phospholipase A1 (PLA1) activity measurements of purified Tle1H in the presence of 1:10 molar ratio of lysozyme from chicken egg white (Lyso) and increasing molar ratio (0, 1, 5, and 10) of SVgrG or SVgrG1-490. Inhibition experiments with SVgrG were repeated three times with independent protein preparations, each measured in triplicate. Inhibition experiments with SVgrG1-490 were repeated twice with two independent protein preparations, each measured in triplicate. Mean values and standard deviation are shown. Statistical analysis relative to the Tle1 activity is indicated. ns, non-significant (P>0.05), *P<0.05, **P<0.01, ***P<0.001, unpaired two-sample Fisher-Pitman permutation test. (P values: Lyso, 0.7006; VgrG 1:1, 0.004946; VgrG 1:5, 1.448e-07; VgrG1:10, 1.448e-07; VgrG1-490 1:1, 0.5627; VgrG1-490 1:5, 0.2977; VgrG1-490 1:10, 0.002631).

C. Representative SDS-PAGE / Coomassie blue staining of the Tle1H, SVgrG and SVgrG1-490 purified proteins used in B (5 μg/well).

Table 1. Interacting amino acids between VgrG and Tle1 as determined by PISA software

VgrG-Tle

(EMD-10218)

(PDB 6SJL)

VgrG

(EMD-10219)

(PDB 6SK0)

Tle-TTR

(EMD-10225)

(PDB 6SKI)

Table 2. Cryo-EM data acquisition and analysis parameters

EXPANDED VIEW FIGURE LEGENDS
PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex (2013)
figf1

(a) Schematic representation of full-length UVRAG with the boundary of the C2 domain indicated above the diagram. C2, phosphoinositide-binding domain; CC, coiled-coil domain. (b) The affinity of the UVRAG-C2 domain for phospholipids was assessed using a protein–lipid overlay assay. The left panel indicates the identity of lipid species on PtdIns strips. Bacterial-purified GST fusion of UVRAG-C2 (wild type, WT), but not GST, binds PtdIns(3)P, PtdIns(4)P and PtdIns(5)P. The K78A/R82A mutant of C2 is defective for phospholipid-binding, whereas the K87A/N88A mutant is partially impaired. Binding of the FYVE and PH domains of Hrs and FAPP1, respectively, to PtdIns(3)P and PtdIns(4)P served as quality controls (right). PE, phosphatidylethanolamine; PC, phosphatidylcholine; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; S1P, sphingosine 1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine. (c) The relative affinity of the GST-fusion proteins for phosphoinositides was quantified by densitometric analysis of the GST immunoblots in b from five independent experiments (n = 5). Error bars represent standard deviation (s.d.). A.U., arbitrary units. (d) Binding of full-length UVRAG (untagged) to lipid blots. The same PtdIns strips (in
b) were used and immunoblotted with antibodies against IgG (right) and UVRAG (left). (e) Multiple sequence alignment of the C2B domain from synaptotagmin-1 (Syt-1) and the C2 domains from phospholipase C beta (PLC- β) and protein kinase C-alpha (PKC- α) and UVRAG, using the ClustalW alignment program. Identical residues are highlighted in red; conserved and similar residues are highlighted in yellow and green, respectively. The two functionally critical residues, Lys 78 and Arg 82, are indicated by an asterisk. The secondary structure of the UVRAG-C2 domain is depicted below the alignment. The sequences of PLC- β, Syt-1 and PKC- α were obtained from Swiss-Prot with accession numbers Q00722, P21707 and P05696, respectively. (f) Homology model of UVRAG-C2 with conserved PtdIns(3)P-interacting residues highlighted with spheres. The crystal structures of PKC- α, PLC- β and Syt-1 C2B were used as templates to develop a homology model of the UVRAG-C2 domain. The binding orientation of PtdIns(3)P with the UVRAG-C2 domain was developed by a molecular docking method and PtdIns(3)P is highlighted in yellow. A close view of the interaction pattern demonstrates that PtdIns(3)P forms hydrogen-bond interactions with the Lys 78 and Arg 82 residue side chains. These two residues are highlighted surrounded by dots and hydrogen bonds are illustrated with black dashed lines. (g) Pulldown of UVRAG-C2 domain GST-fusion proteins by liposomes bearing the indicated phosphoinositides. GST–Hrs-FYVE and GST–FAPP1-PH are also shown.
PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex (2013)
figf2

(a–k) Confocal analyses of the subcellular co-localization of endogenous UVRAG with ER markers, including endogenous calnexin (red, a), endogenous PDI (red, b), and overexpressed DSred-ER (red, c); with Golgi markers, including endogenous GM130 and p115 (red, d,e, cis-Golgi), overexpressed GFP–Man II (green, f, medial-Golgi), endogenous TGN46 (red, g,
trans-Golgi), and overexpressed PtdIns(4)P probe, GFP–FAPP1-PH (green, h); with endogenous coatomer proteins, including Sec31 (red, i, COPII-related) and
β′-COP (red, j, COPI-related); and with the endosome marker GFP-p40phox(PX) (green, k) in HeLa cells. The insets show a high magnification of the selected areas. Scale bars, 10 μm. (l) Confocal microscopic quantification of co-localization of UVRAG with the indicated markers (data are mean±s.d.,
n = 150 cells obtained by gathering data from three independent experiments);
**P0.01;
***P0.001.
PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex (2013)
figf3

(a) UVRAG is released from the ER membrane in cells overexpressing MTMR3. HeLa cells transfected with MTMR3–mStrawberry or the catalytically inactive mutant MTMR3C413S were stained with anti-UVRAG antibody and processed for confocal microscopy. Asterisks indicate MTMR3-transfected cells (left). The percentage of transfected cells with diffused staining of UVRAG was quantified (middle). Data are mean±s.d.;
n = 240 cells collected from four independent experiments;
**P0.01. Western blot analysis shows the levels of endogenous UVRAG in the presence or absence of MTMR3 (right). (b) Wortmannin treatment releases UVRAG from the ER. HeLa cells were treated with 100 nM wortmannin for 1 h, and processed for confocal microscopy using anti-UVRAG and anti-PDI antibodies (top panel). UT, untreated. Insets highlight the relative localization of UVRAG at the ER. Confocal co-localization is indicated (bottom left panel). Data are mean±s.d.;
n = 75 cells for each group from three independent experiments;
***P0.001. Endogenous UVRAG and PDI expression before and after wortmannin treatment were confirmed by immunoblotting (bottom right panel). (c) Mutation of the C2 domain leaves UVRAG unable to associate with the ER. HeLa cells expressing Flag–UVRAG (WT) or its K78A/R82A mutant were fixed and processed for confocal microscopy for co-localization with the ER marker PDI (top panel). Insets highlight the relative distribution of UVRAG at the ER. UVRAG and endogenous PDI expression were confirmed by immunoblotting (bottom panel). See Supplementary Fig. S9 for uncropped data. Scale bars, 10 μm.
PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex (2013)
figf4

(a) The UVRAG C-terminal 270–442 region interacts with RINT-1. Cells (HEK293T) were co-transfected with HA–RINT-1, together with Flag–UVRAG or its mutant derivatives, and whole-cell lysates (WCLs) were immunoprecipitated (IP) with anti-Flag, followed by immunoblotting (IB) with an anti-HA antibody. (b) Schematic representation of UVRAG (wild type, WT) and its deletion mutants, and summary of their interactions with RINT-1. Interaction was determined by co-immunoprecipitation of Flag–UVRAG with HA–RINT-1 from HEK293T cell lysates. +, strong binding; −, no binding. (c) Interaction between endogenous UVRAG and RINT-1. WCLs of HEK293T cells were used for immunoprecipitation with control serum (control) or an anti-UVRAG (left) or anti-RINT-1 antibody (right), followed by immunoblotting with the indicated antibodies. The bottom panel shows endogenous protein expression. (d) ER localization of RINT-1, ZW10 and UVRAG. HeLa cells were stained with anti-UVRAG (green), anti-PDI (ER marker, red), and anti-ZW10 or anti-RINT-1 (blue), followed by confocal microscopy. Insets highlight UVRAG co-localization with RINT-1 and ZW10 at the ER. Scale bars, 10 μm. (e) Interaction between endogenous UVRAG and the RINT-1–ZW10–NAG tethering complex proteins. WCLs of HEK293T cells were used for immunoprecipitation with control serum (IgG) or an anti-UVRAG (first panel), anti-RINT-1 (second panel), anti-ZW10 (third panel) and anti-NAG (fourth panel), followed by immunoblotting with the indicated antibodies. As input, 10% of the WCLs was used. Syn 18, syntaxin 18; Syn 6, syntaxin 6. See Supplementary Fig. S9 for uncropped data.