EMBO/SourceData huggingface dataset:
Figure legend: "(A,B) HEK 293T cells were transfected with HA-Akt (A) or Myc-RSK (B), and/or Flag-ATG1α WT or KI plasmids. The immunoprecipitated Akt and RSK proteins were analysed by immunoblot using appropriate antibodies (top three panels). Flag (ATG1α) blots were completed from the same cell lysates (bottom panel)."
NCBI gene IDs:
"Akt: 207
RSK: 6195///6197///6196
ATG1α: 8408"
Figure legend: "(C) HEK 293T cells were transfected with HA-S6K Thr 389 Glu mutant and/or Flag-ATG1α WT or KI. The immunoprecipitated S6K mutants were analysed by immunoblot using anti‐phosphospecific S6K Thr 229 (top panel) and HA (S6K) antibodies (middle panel). Flag (ATG1α) blot was completed from the same cell lysates (bottom panel). ATG1, Autophagy‐specific gene 1; EGF, epidermal growth factor; HA, haemagglutinin; KI, ATG1α Lys 46 Ile (kinase‐dead) mutant; RSK, ribosomal S6 kinase; WT, wild type."
NCBI gene IDs:
"ATG1α: 8408"
Figure legend: "b, Fluorescence microscopy of HeLa cells transfected with either IpaJ or VirA. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 µm."
NCBI gene IDs:
"IpaJ: 876675
VirA: 876471"
Figure legend: "b, Fluorescence microscopy of HeLa cells transfected with the indicated IpaJ mutants. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 µm."
NCBI gene IDs:
"IpaJ: 876675"
Figure legend: "c, IpaJ or its catalytic mutants were expressed from a galactose-inducible promoter (pGal413 vector) and assayed for a growth arrest phenotype on galactose or glucose (control) carbon source."
NCBI gene IDs:
"Gal413: 
IpaJ: 876675"
Figure legend: "e, Yeast strain harbouring a galactose-inducible IpaJ gene were transformed with a multi-copy vector containing the indicated genes. Yeast were assayed for a growth arrest phenotype on galactose or glucose (control) carbon source."
NCBI gene IDs:
"IpaJ: 876675"
Figure legend: "a-c, Mass spectra of purified ARF1-strep in untreated (a), IpaJ-treated (b) or IpaJ C64A-treated (c) cells, with the observed monoisotopic mass reported in daltons. *Observed 1 Da shift in molecular mass is accounted for in MS/MS data (Supplementary Fig. 7)."
NCBI gene IDs:
"IpaJ: 876675"
Figure legend: "f, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-His isolated from bacteria either not expressing (lane 1) or expressing (lane 2) NMTp. Bacterial cell lysates treated with recombinant IpaJ or IpaJ C64A as indicated. The expression levels of ARF1-His were determined by Coomassie blue stain."
NCBI gene IDs:
"IpaJ: 876675
NMTp: 850892"
Figure legend: "g, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-strep purified from HeLa cell lysates left untreated or incubated with IpaJ or its catalytic mutant as indicated. The expressed amounts of ARF1-strep are shown (bottom panel)."
NCBI gene IDs:
"IpaJ: 876675"
Figure legend: "h, In-gel fluorescence assay visualizing protein extracts isolated from HeLa cells incubated with azide myristic acid, azide palmitic acid or geranylgeranyl alcohol azide and subsequently labelled with Alexa Fluor 647 alkyne by click chemistry. Arrows indicate proteins that are proteolytically demyristoylated by IpaJ."
NCBI gene IDs:
"IpaJ: 876675"
Figure legend: "b, In-gel fluorescence assay showing myristoylated peptides of the indicated proteins in cells expressing IpaJ or catalytic mutant. The peptide-eGFP and IpaJ inputs are indicated. *Demyristoylation of GRASP65 and hVPS15 resulted in slower mobility of the resulting peptide, potentially owing to reduced mobility in SDS-polyacrylamide gel electrophoresis caused by proteolytic reaction."
NCBI gene IDs:
"IpaJ: 876675"
Figure legend: "c, In-gel fluorescence assay showing myristoylated ARF1 T31N mutant (a GDP-locked mutant) or ARF1 Q71L (a GTP-locked mutant) after in cells co-expressing IpaJ or catalytic mutants as indicated. Equal loading of ARF1 (middle panel) and IpaJ (lower panel) was determined by western blot analysis."
NCBI gene IDs:
"ARF1: 375
IpaJ: 876675"
Figure legend: "d, Fluorescence microscopy showing ARF1-mCherry and the Golgi apparatus (GM130) after cellular microinjection of recombinant IpaJ or IpaJ C64A, over the indicated period. Scale bar, 10 µm."
NCBI gene IDs:
"IpaJ: 876675"
Figure legend: "Detection of native and GFP-fused TgAtg8.Protein extracts corresponding to 107 tachyzoites from parental RHΔHX and transgenic GFP-TgAtg8 cell lines were separated by SDS-PAGE and analysed by Western blot using anti-TgAtg8 or anti-GFP antibodies. Overexpressed GFP-TgAtg8 and native TgAtg8 are indicated by arrows."
NCBI gene IDs:
"Atg8: 7900047"
Figure legend: "A. Protein extracts corresponding to tachyzoites incubated in the same starvation conditions as in Figure 2B were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect TgAtg8 and the lipidated TgAtg8-PE form. GFP-TgAtg8 parasites extracts were analysed with anti GFP antibody (left), while parental cell line was analysed with anti-TgAtg8 antibody (right). Anti-ROP5 was used as a loading control."
NCBI gene IDs:
"Atg8: 7900047"
Figure legend: "B. Cell lysates from GFP-TgAtg8 parasites were subjected to a centrifugation at 100,000 g to separate the soluble fraction (high speed supernatant, HSS) from the membrane fraction (high speed pellet, HSP). The faster migrating form of GFP-TgAtg8 is exclusively present in the membrane fraction as revealed by Western blot analysis after urea SDS-PAGE using anti-GFP"
NCBI gene IDs:
"Atg8: 7900047"
Figure legend: "D. GFP-TgAtg8-expressing parasites were grown in host cells in the presence of 3H-ethanolamine and then starved in HBSS for 8 hours, still in the presence of the radioactive PE precursor. Tachyzoites were then lysed and GFP-TgAtg8 was immunoprecipitated using anti-TgAtg8 antibody and analysed by urea SDS-PAGE. The immunoprecipitated forms of GFP-TgAtg8 were detected by Western blot using anti-GFP antibody and radioactive ethanolamine was found to be incorporated into immunoprecipitated GFP-TgAtg8, as detected by fluorography (3H-etn)."
NCBI gene IDs:
"Atg8: 7900047"
Figure legend: "A. Fluorescence microscopy analysis of extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version, before or after induction of autophagy for 8 hours in HBSS. Autophagosomes are labelled by GFP-TgAtg8 in control parasites (arrowheads), but not by the glycine mutant version of the protein. B. Extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version were put to starve in HBSS medium for increased lengths of time and the proportions of cells displaying punctate or cytosolic GFP signals were assessed. Data are mean from n = 4 independent experiments ±SEM."
NCBI gene IDs:
"Atg8: 7900047"
Figure legend: "C. Protein extracts corresponding to tachyzoites expressing GFP-TgAtg8 and glycine mutant version incubated in HBSS for up to 8 hours, were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect GFP-TgAtg8 and lipidated GFP-TgAtg8-PE forms. Anti-ROP5 was used as a loading control."
NCBI gene IDs:
"Atg8: 7900047"
Figure legend: "B. Fluorescence microscopy pictures of GFP-TgAtg8-expressing tachyzoites during their intracellular development and division. The arrowhead indicates the residual body made of maternal material left after endodyogeny."
NCBI gene IDs:
"Atg8: 7900047"
Figure legend: "A. Schematic representation of the strategy. Recombineered cosmid bearing chloramphenicol acetyl transferase (CAT) gene for selection of resistant parasites, flanked by homologous regions from TgAtg3, was used to replace endogenous genomic locus in the cell line expressing a regulatable extra copy of TgAtg3 (imyc-sTgAtg3). Mutants obtained after double homologous recombination events were selected with chloramphenicol. PCR amplifications and Southern blot strategy used for subsequent characterisation of the mutant are indicated in red and green, respectively. B. PCR verification of the removal of TgAtg3 in the mutant (imyc-sTgAtg3-ΔAtg3, A) compared to the parental cell line (imyc-sTgAtg3, B)"
NCBI gene IDs:
"Atg3: 7900125"
Figure legend: "C. Southern blot verification of the TgAtg3 gene replacement. ∼4 µg of genomic DNA from mutant (A) and parental (B) tachyzoites were digested by HindIII/AvrII, transferred on a nylon membrane and probed with 32P-labeled 5′ and 3′ DNA probes located as schematised in A."
NCBI gene IDs:
"Atg3: 7900125"
Figure legend: "A. The vector for the expression of the ATc-regulatable sTgAtg3 extra copy allows myc-epitope tagging of this protein. Anti-myc detection of myc-tagged TgAtg3 protein in intracellular parasites either without treatment with ATc, or with a continuous 2 days treatment with 1.5 µg/ml ATc, or after reinvasion following a continuous 4 days treatment. DIC: differential interference contrast."
NCBI gene IDs:
"Atg3: B9Q737///7900125"
Figure legend: "C. Western blot analysis following urea SDS-PAGE of TgAtg3-depleted parasites extracts, showing an absence of upregulation of the autophasome-bound TgAtg8-PE form. Anti-ROP5 was used as a loading control."
NCBI gene IDs:
"Atg3: 7900125"
Figure legend: "A. Confluent monolayers of fibroblasts were infected either with extra copy-expressing imyc-sTgAtg3 parental cell line in the presence of ATc (a), imyc-sTgAtg3-ΔAtg3 mutant cell line without ATc (b), mutant cell line in the presence of ATc (c), or mutant cell line pre-incubated with ATc before start of the experiment and maintained in the presence of ATc. Plaques (arrowheads) resulting from the lysis of host cells due to the multiplication of the parasites are only visible when TgAtg3 is still expressed. B. Mean plaque area comparisons between fibroblast layers infected with controls and TgAtg3-depleted parasites (a, b, c: legend as in A). Plaques observed with the mutant in the presence of ATc were significantly smaller than with control cell lines (* p&lt;0.005, Student's T test). Data are mean from n = 3 independent experiments ±SEM."
NCBI gene IDs:
"Atg3: 7900125"
Figure legend: "C. Numbers of parasites per vacuole are significantly lower in TgAtg3-depleted cell line (pre-incubated for 4 days with ATc) compared with controls 24 or 48 hours post invasion. Data are mean from n = 3 independent experiments ±SEM."
NCBI gene IDs:
"Atg3: 7900125"
Figure legend: "TgAtg3-depleted parasites show a defect in mitochondrion morphology.Mitochondrion labelled with specific antibodies (see results section) was found as a reduced structure, or even absent, in TgAtg3-depleted cell lines (middle series of micrographs, kept for 2 days in the presence of ATc or pre-incubated with ATc for 4 days before invasion, respectively). Parasites left to develop for two days in the presence of ATc to progressively extinguish the expression of TgAtg3 showed an accumulation of mitochondrial marker at the residual body. TgAtg3 depletion was verified by detecting myc-tagged regulatable extra-copy with specific antibody. TgAtg3-expressing cell line imyc-sTgAtg3cultivated in the presence of ATc for 4 days was used as a control for mitochondrial morphology (bottom). DNA was labelled with DAPI."
NCBI gene IDs:
"Atg3: 7900125"
Figure legend: "Altered mitochondrial ultrastructure in TgAtg3-depleted tachyzoites observed by electron microscopy.Ultrathin section of a TgAtg3-depleted (3 days of ATc treatment) tachyzoite showing the dramatic alteration of the mitochondrion, where cristae remnants are still visible (arrows), but which is filled up with membranous profiles (arrowheads), whereas Golgi (G), apicoplast (A), rhoptries (R) and dense granule (D) look normal."
NCBI gene IDs:
"Atg3: 7900125"
Figure legend: "(B) SCPO, STIPO and STAPO after indicated siRNA treatment of GFP-LC3‐HEK cells incubated as in (A). Error bars represent s.e.m. Significance was determined using a two‐tailed paired t‐test compared with RISCfree (RF) in EL: SCPO siULK1, ***P=0.0002; siATG7, **P=0.0078; siNRBP2, **P=0.0088. STIPO siULK1, ***P0.0001; siATG7, **P=0.0060; siNRBP2, **P=0.0041. STAPO siULK1, ***P0.0001; siATG7, **P=0.0037; siNRBP2, **P=0.0028. The experiments were performed: RISCfree (n=5), siULK1 (n=5), siATG7 (n=3), siNRBP2 (n=3)."
NCBI gene IDs:
"ATG7: 10533
NRBP2: 340371
ULK1: 8408"
Figure legend: "(D) Scatter plot of median SCPO Z‐scores from siRNA pools in the primary screen that met the minimum object count (OC) cutoff. Dashed lines represent approximate cutoff for 500 best increasers and decreasers. RP of known autophagy genes (ATG4C, ATG14, RB1CC1, ATG9A, ATG7) are given in Supplementary Table S2; AMBRA1 (FLJ20294), TBC1D25 (OATL1) and SH3GLB1 (BIF‐1) in Supplementary Table S1; LAMTOR2 has a RP=708.7 and RUBICON has a RP=1126."
NCBI gene IDs:
"AMBRA1: 55626
ATG14: 22863
ATG4C: 84938
ATG7: 10533
ATG9A: 79065
RUBICON: 9711
LAMTOR2: 28956
RB1CC1: 9821
SH3GLB1: 51100
TBC1D25: 4943"
Figure legend: "(C-E) STIPO results after incubation under different autophagy‐inducing conditions following (C) SCOC knockdown (D) SUPT5H knockdown and (E) WAC knockdown. siRNA‐treated cells were incubated for the indicated time in FL: full medium plus leupeptin; ES; EL: ES plus leupeptin; Torin: 250 nM Torin1 in FL; Resv: 128 μM Resveratrol in FL; LiCl: 10 mM LiCl in FM."
NCBI gene IDs:
"SCOC: 60592
SUPT5H: 6829
WAC: 51322"
Figure legend: "(C) Endogenous SCOC (green top, red bottom) partially colocalises with endogenous LC3 (red, top) and GFP-LC3 (green, bottom) in HEK293 and GFP-LC3‐HEK cells, respectively, after 2 h amino‐acid starvation. Boxed region is at higher magnification in merge panel on right. Scale bars=10 μm."
NCBI gene IDs:
"LC3: 440738///81631///84557"
Figure legend: "(A) SCOC is required for autophagy in HEK293 cells. Anti‐ULK1, ‐Actin and ‐LC3 blot after siRNA treatment in HEK293 cells in FM, ES or EL. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RF EL versus siSCOC‐03 EL, *P=0.0419."
NCBI gene IDs:
"SCOC: 60592"
Figure legend: "(B) SCOC is required for p62 degradation. p62 levels were determined by western blot (Supplementary Figure S5) after incubation in FM, ES for 2 or 4 h, or ES with BafilomycinA1 (ES+BAF) for 2 and 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree ES 2 and 4 h, ***P&gt;0.0001 and **0.0014, respectively."
NCBI gene IDs:
"SCOC: 60592"
Figure legend: "(C) SCOC-GFP and SCOC-myc levels are reduced by SCOC siRNA duplex‐03. Anti‐GFP, ‐Myc and ‐Actin blots of HEK293 cells after treatment with ULK1, RISCfree or SCOC siRNA and overexpression of GFP, SCOC-myc or SCOC-GFP."
NCBI gene IDs:
"SCOC: 60592
ULK1: 8408"
Figure legend: "(D) Overexpression of SCOC-myc increases autophagy. Anti‐Actin, ‐LC3 and ‐Myc blots of HEK293 cells after transfection of pcDNA or SCOC-myc. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=5). SCOC-myc EL versus pcDNA EL, *P=0.0215."
NCBI gene IDs:
"SCOC: 60592"
Figure legend: "(A) Knockdown of FEZ1 enhances LC3 lipidation but does not affect p62 degradation. Anti‐Actin and ‐LC3 blot after siRNA treatment in HEK293 cells. Quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RISCfree ES versus siFEZ1‐01 ES, P=0.034. Quantification of p62/actin ( Supplementary Figure S5) performed as in Figure 4B; error bars represent s.e.m. (n=3); RISCfree FM versus RISCfree 2 and 4 hP&gt;0.0001 and P=0.0014, respectively. qRT-PCR showing FEZ1 mRNA levels after indicated knockdown in GFP-LC3‐HEK cells (error bars represent s.d.)."
NCBI gene IDs:
"FEZ1: 9638
LC3: 440738///81631///84557"
Figure legend: "(B) Overexpression of FEZ1 inhibits LC3 lipidation and p62 degradation. Anti‐Myc, ‐Actin and ‐LC3 blot after transfection with pcDNA or Myc-FEZ1 in HEK293 cells. Quantification of LC3II/actin; error bars represent s.e.m. (n=3); pcDNA ES versus Myc-FEZ1 ES, P=0.0318. Anti‐GFP, ‐Actin and ‐p62 blot after transfection with GFP or FEZ1-GFP and treatment with ES or ES plus Bafilomycin (EB) for 4 h. Quantification of averaged duplicates; error bars represent s.e.m. (n=3); GFP FM versus GFP ES, P=0.0077; GFP FM versus GFP EB, P=0.0212; GFP ES versus FEZ1-GFP ES, P=0.048."
NCBI gene IDs:
"FEZ1: 9638"
Figure legend: "(E) The FEZ1-SCOC interaction requires the evolutionary conserved residues L254 and L260 in the coiled‐coil domain of FEZ1 as demonstrated in GST‐pulldown assays with in vitro translated 35S‐labelled WT or mutant Myc‐FEZ1 and GST or GST‐SCOC. Autoradiographs of 10% input and bound proteins (top panels) and Coomassie blue‐stained gels of bead‐bound GST and GST‐SCOC proteins used (bottom panels)."
NCBI gene IDs:
"FEZ1: 9638"
Figure legend: "(A) FEZ1 interacts with both the kinase domain (1-278) and the proline-serine rich spacer of ULK1 (279-828). GFP‐ or Myc‐tagged ULK1 fragments were in vitro translated with 35S‐methionine and subjected to GST‐pulldown assays using GST and GST-FEZ1 purified from E. coli (bottom). Bound proteins were detected by autoradiography following SDS-PAGE (top)."
NCBI gene IDs:
"FEZ1: 9638
ULK1: 8408"
Figure legend: "(C) The FEZ1-GFP and Myc-ULK1 interaction is unaffected by amino‐acid starvation. Anti‐Myc and ‐GFP blots after HEK293 cells were co‐transfected with FEZ1-GFP and Myc-ULK1, incubated in FM or ES for 2 h, harvested and the complex was immunoprecipitated using anti‐GFP antibody."
NCBI gene IDs:
"FEZ1: 9638
ULK1: 8408"
Figure legend: "(D) BN-PAGE reveals SCOC and FEZ1 are in a complex. FEZ1-GFP, FLAG-SCOC and Myc-ULK1 wild type or kinase‐inactive ULK1K46I (ULK1KI) were co‐expressed in HEK293 cells. Anti‐GFP, ‐FLAG and ‐Myc western blots are shown for the BN-PAGE gel (top) or the SDS-PAGE gel (bottom). In GFP and FLAG western blots, two different exposures of the same BN-PAGE blot are shown spliced together."
NCBI gene IDs:
"ULK1: 8408"
Figure legend: "(E) siRNA depletion of ULK1 does not affect the interaction of FEZ1 with SCOC. RF or ULK1 siRNA‐treated cells were co‐transfected with FEZ1-GFP and FLAG-SCOC, then incubated and immunoprecipitated as in (C). Western blot of lysates for ULK1, GFP and FLAG, and immunoprecipitates with GFP and FLAG antibodies."
NCBI gene IDs:
"ULK1: 8408"
Figure legend: "(F) siRNA depletion of SCOC reduces FEZ1-ULK1 complex. HEK293 cells treated with either SCOC or ULK1 siRNAs were transfected with FEZ1-GFP. FEZ1-GFP was detected with anti‐GFP in both BN-PAGE and SDS-PAGE. Endogenous ULK1 was detected with anti‐ULK1 antibody. Arrowheads indicate a FEZ1-GFP and endogenous ULK1 complex at 200 kDa and a larger MW complex &gt;1236 kDa also detected in (D) in the triple transfection. The 200 KDa complex is sensitive to loss of SCOC and ULK1."
NCBI gene IDs:
"SCOC: 60592
ULK1: 8408"
Figure legend: "(A) SCOC interacts with UVRAG. HEK293 cells were transfected with FLAG-SCOC and Myc-UVRAG, lysed and incubated with anti‐FLAG (left) or anti‐Myc (right) antibodies. Co‐immunoprecipitation of Myc-UVRAG (left) and FLAG-SCOC (right) was confirmed using anti‐Myc and anti‐FLAG antibodies for western blotting. Asterisk in (A-C) and (D) indicates non‐specific bands."
NCBI gene IDs:
"SCOC: 60592
UVRAG: 7405"
Figure legend: "(B) Interaction of SCOC with UVRAG is unaffected by FEZ1-GFP. As in (A), co‐immunoprecipitation was preformed in HEK293 cells transfected with FLAG-SCOC, Myc-UVRAG and FEZ1-GFP. Myc-UVRAG was detected as in (A), FEZ1-GFP was detected with anti‐GFP antibody."
NCBI gene IDs:
"FEZ1: 9638
SCOC: 60592
UVRAG: 7405"
Figure legend: "(C) The SCOC and UVRAG interaction is reduced by starvation, and this reduction is inhibited by FEZ1-GFP. HEK293 cells were transfected as in (B) and 24 h later either treated with FM or ES for 2 h. FLAG-SCOC or Myc-UVRAG was immunoprecipitated with anti‐FLAG or anti‐Myc antibodies and the bound proteins were detected with anti‐Myc, ‐GFP or ‐FLAG antibodies by western blotting. Quantification of the amount of FLAG-SCOC co‐immunoprecipitated with Myc-UVRAG in FM or ES, without overexpressed FEZ1-GFP (left) or with overexpressed FEZ1-GFP (right). Significance was determined using a two‐tailed paired t‐test (n=3)."
NCBI gene IDs:
"FEZ1: 9638
UVRAG: 7405"
Figure legend: "(D) FEZ1 interacts directly with endogenous UVRAG and is required for SCOC interaction. HEK293 cells were transfected with FEZ1-GFP, GFP or FLAG-SCOC alone or in combination. Top panel input, bottom panel immunoprecipitation with anti‐GFP or anti‐FLAG. Western blot for endogenous UVRAG, GFP-FEZ1 GFP or FLAG-SCOC as indicated. Arrowhead: endogenous UVRAG."
NCBI gene IDs:
"FEZ1: 9638
SCOC: 60592"
Figure legend: "(A) Depletion of endogenous WAC. Anti‐WAC and ‐Actin blot and WAC mRNA levels by qRT-PCR after treatment with RF or individual WAC siRNAs (W‐01 to W‐04) in HEK293 cells. qRT-PCR error bars represent standard deviation of triplicates (n=2)."
NCBI gene IDs:
"WAC: 51322"
Figure legend: "(B) (B) WAC is required for autophagy. Anti‐ULK1, ‐WAC, ‐Actin and ‐LC3 blots after siRNA treatment in HEK293 cells and incubation in FM, ES or EL. Representative blot; quantification of LC3II/actin of averaged duplicates; error bars represent s.e.m. (n=3). Significance was determined using a two‐tailed paired t‐test: RF EL versus siWAC‐03 EL, **P=0.0079."
NCBI gene IDs:
"WAC: 51322"
Figure legend: "(C) p62 degradation requires WAC. Anti‐p62 and ‐Actin blot after siRNA treatment of GFP-LC3‐HEK cells in FM or ES for 2 h in duplicate. Bars represent p62/actin levels normalised to RF FM; quantification of p62/actin; error bars represent s.e.m. (n=3): RF FM versus RF ES, **P=0.0057; RF FM versus siWAC‐03 FM, **P=0.0025; RF ES versus siWAC‐03 ES, *P=0.0386."
NCBI gene IDs:
"LC3: 440738///81631///84557
WAC: 51322"
Figure legend: "(D) WAC depletion accelerates degradation of a UPS reporter. Anti‐p62, ‐GFP, ‐Actin, ‐LC3, ‐WAC and ‐Atg16 blots after siRNA treatment in UbG76V-YFP MelJuSo cells and incubation conditions FM, ES or FM with 10 μM MG132 (MG) for 2 h. Quantification of normalised average GFP intensity in FM of RISCfree compared with WAC‐03 siRNA treatment (n=3) and compared with ATG16 siRNA treatment (n=2). Error bars represent s.e.m.: RF FM versus siWAC‐03 FM, **P=0.0082."
NCBI gene IDs:
"ATG16: 89849///55054
WAC: 51322"
Figure legend: "(E) Soluble HttQ25-CFP degradation is increased after WAC depletion. Anti‐WAC, ‐Actin and ‐GFP blots after indicated siRNA treatment (siWAC duplexes ‐02, ‐03, ‐04) of HttPolyQ‐mutant HeLa cell lines and incubation conditions DMSO or DOX (Doxycyline) for 3 days. The insoluble fraction was obtained by pelleting the HttQ103 lysates (see Materials and methods). Quantification of normalised average HttQ25-CFP intensity in DMSO of RISCfree compared with WAC siRNA treatment; error bar represents s.e.m. (n=3): HttQ25-CFP/actin RF DMSO versus siWAC DMSO, *P=0.0350; soluble HttQ103-CFP/actin RF DMSO versus siWAC DMSO, *P=0.0380."
NCBI gene IDs:
"Htt: 3064
WAC: 51322"
Figure legend: "(A) Western blot analysis of FADD, FLIP and Caspase 8 at the TRAIL-R2 DISC in HCT116 cells treated with escalating doses (0-30nM) of caspase-8 siRNA for 48 hours. Samples were incubated with anti-TRAIL-R2 (AMG655) beads (4x) for 90 minutes prior to collection. A scrambled control siRNA (SC) was transfected (30nM) for comparison. (B) Quantification of Caspase 8 (p55, p43/41 and p26/24) and FLIP (FLIP(L), FLIP(S) and p43-FLIP) at the TRAIL-R2 DISC by densitometry and normalised to known protein standards (C) Ratio of Caspase-8:FLIP calculated from values presented in (B)."
NCBI gene IDs:
"caspase-8: 841"
Figure legend: "(E) Western blot analysis of FLIP, Caspase 8, and FADD recruitment to the TRAIL-R2 DISC in PC3 cells stably expressing an empty vector (EV) or flag tagged FLIP L (FL) treated with 2.5µM MS-275 for 48 hours, followed by a 90 minute DISC IP. * modified form of FLIP, potentially mono-ubiquitinated p43-FLIP(L)."
NCBI gene IDs:
"flag: 
FLIP: 8837"
Figure legend: "(A) Western Blot analysis of Caspase 8, Caspase 10, FLIP and FADD recruitment to the TRAIL-R2 DISC in procaspase 8 (WT) or procaspase 8 deficient (Null) A549 cells incubated with AMG655 beads (4x) for either 30, 60 or 180 minutes."
NCBI gene IDs:
"procaspase 8: 841"