Journal: Cell Death and Differentiation
Article Title: Autophagy induced during apoptosis degrades mitochondria and inhibits type I interferon secretion
Figure Lengend Snippet: Bax/Bak-activation activates ULK1 via AMPK a Schematic of the regulation of autophagy protein ULK1. AMPK activates ULK1 by phosphorylating both S317 and S555 on ULK1, while mTORC1 inhibits ULK1 activity by phosphorylating S757. b Apaf-1 −/− MEFs with Mcl-1 deleted by CRISPR-Cas9 technology were treated with 1 μM ABT-737, 50 μM CCCP, or starved in HBSS for 4 h prior to lysis and western blotting. Identical aliquots of samples were run on four gels as follows: #1 (AMPK, p-ULK1 S757 ), #2 (p-AMPK T172 , p-ULK1 S317 , S6, β-actin), #3 (p-ULK1 S555 , 4E-BP1), #4 (p-S6 S240/244 , ULK1, p-4E-BP1 S65 , LC3B). c Apaf-1 −/− MEFs were treated with 1 μg/mL Dox to express Bim s , 50 μM CCCP, or starved in HBSS for 4 h prior to lysis and western blotting. Identical aliquots of samples were run on four gels as follows: #1 (LC3B, p-ULK1 S757 ), #2 (p-AMPK T172 , p-ULK1 S317 , 4E-BP, S6), #3 (p-4E-BP1 S65 , ULK1, AMPK), #4 (p-S6 S240/244 , p-ULK1 S555 . d AMPK activation is upstream of LC3B lipidation. Mcl-1 −/− MEFs were infected with two different guide RNAs against ATG5 and selected for puromycin resistance. Pools were then treated with 1 μM ABT-737 for 4 h prior to western blotting. Identical amounts of sample were loaded on four different gels. e The intracellular ratio of AMP:ATP increases upon Bax/Bak-activation. MEFs were treated with either 1 μM ABT-737 or 1 μg/mL Dox to express Bim s for 4 h and analysed by mass spectrometry. The mean of five to six experiments is shown; error bars represent the SEM
Article Snippet: Equal amounts of protein were separated on 10% NUPAGE® Bis Tris gels (Invitrogen), transferred to nitrocellulose, and blotted with the antibodies raised against Mcl-1 (19C4; WEHI), Bim (3C5; WEHI), Bax (49F9-13-13; WEHI), Bak (Sigma), Parkin (sc-32282; Santa Cruz), LAMP1 (1D4B; Santa Cruz), TNFR1 (ab19139; Abcam), Calnexin (ab22595; Abcam), Apaf-1 (18H2; WEHI), TIMM44 (HPA043052; Sigma) and β-actin (AC-15; Sigma).
Techniques: Activation Assay, Activity Assay, CRISPR, Lysis, Western Blot, Infection, Mass Spectrometry