Fig 1: ATG3 protein is a target of LC3/GABARAP conjugation and ATG4-mediated deconjugation. A, enrichment of transiently expressed 3xFLAG–LC3B G120 from HeLa control and ATG4B KO cells by IP with anti-FLAG antibody prior to Western blotting with anti-FLAG antibody (left) and anti-ATG3 antibodies (middle, right panel). Cells were transfected with GFP as a negative control. B, 3xFLAG–LC3B G120 expressed in HeLa ATG4B KO cells was enriched by IP with anti-FLAG antibody prior to treatment with 0.02 mg/ml recombinant GST–ATG4B WT or C74S at 37 °C for 1 h and Western blotting using anti-FLAG and anti-ATG3 N-ter antibody. C, HeLa control and ATG4B KO cells were transfected with 3xFLAG–LC3B G120, 3xFLAG–LC3B Q116P G120, or GFP as a negative control prior to harvesting. Western blotting was performed to detect 3xFLAG–LC3B and ATG3 using the same lysates diluted with either nonreducing (β-mercaptoethanol excluded) or reducing (standard recipe) sample buffer. Thioester-linked species are detected under nonreducing conditions only. D, Western blotting of HeLa ATG4B KO GFP–LC3B G120 cells transiently transfected with 3xFLAG–ATG3 WT and point mutant constructs. Samples were lysed in buffer lacking NEM and processed under reducing conditions (upper panel). The same constructs were used to rescue HeLa ATG3 KO cells treated for 3 h with 250 nm Torin1 and 10 nm baf A1 and assessed by Western blotting under nonreducing conditions (lower panel). E, schematic of human ATG4 protease cellular function in protein deconjugation (deLC3ylation), which counteracts covalent attachment of LC3/GABARAP (LC3ylation) to ATG3 protein at residue Lys-243.
Fig 2: Silencing Huwe1 induces the expression of autophagy-related proteins under OGD/R conditions. Huwe1 shRNA lentivirus was infected into cortical neuronal cells on DIV 3, and then the cells were subjected to OGD for 3 h and reperfusion for 24 h on DIV 7. (A) Huwe1 shRNA lentivirus decreased the mRNA expression of Huwe1 at 24 h after reperfusion, as measuring via reverse transcription-quantitative PCR, with GAPDH as an internal control. (B) Huwe1 shRNA decreased the protein expression levels of (C) Huwe1 and ubiquitin at 24 h after reperfusion, but significantly increased the expression of (D) autophagy-related proteins (Beclin-1, ATG7, ATG5, ATG3, LC3 and p62) compared with the V-ctrl group, as detected using western blotting. Tubulin was used as an internal control for western blotting. All experiments were repeated three times independently. #P<0.05 vs. the control group; ∆P<0.05 vs. the V-ctrl group. DIV, days in vitro; V-ctrl, green fluorescent protein-scramble control lentivirus-treated group; R, reperfusion; OGD, Oxygen-glucose deprivation; sh, short hairpin RNA; Huwe1, HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1; ATG, autophagy related; LC3, microtubule associated protein 1 light chain 3 α.
Fig 3: Characterization of human ATG3, ATG5 and ATG7 knockout cell lines. (A) Western blot validation of CRISPR/Cas9-generated ATG3, ATG5 and ATG7 KO HeLa cell clones following culturing in complete medium, using antibodies targeting ATG3, ATG5 and ATG7 protein. p62/SQSTM1 and LC3B were also detected to assess autophagy phenotypes. Vinculin was used as a loading control. (B) Western blot analysis of the effect of autophagy inducer (Torin1, 250 nM) and inhibitor (baf A1; bafilomycin A1, 10 nM) single and combined treatments (for 3 hours prior to lysis) of HAP1 control and ATG5 KO cells. Lysates were probed with antibodies against p62/SQSTM1 and LC3B, as well as ATG5 to detect ATG12-ATG5 conjugate. † denotes band of unknown identity which may correspond to the LC3B-AMP intermediate. Actin was used as a loading control. (C) HAP1 control and ATG5 KO cells treated with Bafilomycin A1 (10 nM) or Torin1 (250 nM) for 3 hours were lysed, and samples were prepared for western blotting in the presence (reducing) or absence (non-reducing) of β-mercaptoethanol in the sample buffer. Membranes were probed with an antibody against ATG3 to assess total ATG3 expression and the redox-sensitive thioester linkage of LC3B with ATG3. An antibody against LC3A/B was used to visualize LC3B linked via thioester bond with ATG3 and ATG7 (visible only under non-reducing conditions) and LC3B lipidation. † denotes band of unknown identity which may correspond to the LC3B-AMP intermediate. Vinculin was detected as a loading control.
Fig 4: ATG5 is dispensable for protein ATG8ylation. (A) HAP1 control and ATG5 KO cells transfected with 3xFLAG-LC3B G120 or 3xFLAG-LC3B Q116P G120 were treated with DMSO or 250 nM Torin1 and 10 nM bafilomycin A1 for 3 hours prior to lysis. Lysates were analyzed by western blotting using antibodies against the FLAG tag, ATG5 (to detect ATG12-ATG5 conjugate) or Vinculin (as a loading control). (B) HeLa control, ATG3 KO, ATG5 KO or ATG7 KO cells were transfected with Flag-tagged LC3B G120 or LC3B Q116P G120 and treated for 3 hours with DMSO or or 250 nM Torin1 and 10 nM bafilomycin A1 prior to lysis. western blotting of lysates was performed and the membrane was probed with antibodies against FLAG tag, or Actin as a loading control.
Supplier Page from Abcam for Anti-ATG3 antibody [EPR4802]