Extended Data Fig. 3: Beclin 1 is palmitoylated by DHHC5.
From: Reduction of DHHC5-mediated beclin 1 S-palmitoylation underlies autophagy decline in aging
a, HEK293T cells expressing Flag-Beclin 1 were incubated with 17-ODYA or palmitic acid (PA) overnight followed by immunoprecipitation and Cu(I)-assisted cross linking to TAMRA-azide and the immunoprecipitated samples were then resolved by SDS-PAGE and scanned to record the in-gel fluorescence signal. After scanning, the gel was stained with Coomassie Brilliant Blue (CBB) for protein loading. b, Schematic of the immunoprecipitation and acyl-biotin exchange (IP-ABE) assay followed by streptavidin pulldown to purify and detect Beclin 1 palmitoylation. c, HEK293T cells expressing Flag-Beclin 1 were subjected to IP-ABE assay to detect Palm-Beclin 1. d, e, HEK293T cells expressing Flag-Beclin 1 were treated with 200 nM Torin-1 (d) or starved in EBSS (e) for indicated time. The cell lysates were then subjected to IP-ABE assay to detect Palm-Beclin 1. Quantification of Palm-Beclin 1 normalized to total Beclin 1 is relative to untreated control for each independent experiment. f, HEK293T cells expressing Flag-Beclin 1 and indicated DHHCs were subjected to IP-ABE assay to detect Palm-Beclin 1. Quantification of Palm-Beclin 1 normalized to total Beclin 1 is relative to no DHHC control. g, WT and indicated DHHC-KD H4 cells expressing Flag-Beclin 1 were subjected to IP-ABE assay to detect Palm-Beclin 1. The knockdown efficiency of DHHCs was confirmed by quantitative PCR. h, DHHC5-WT/KD HEK293T cells were transfected with Flag-Beclin 1 for 24 h. The cell lysates were then subjected to IP-ABE assay with or without HAM treatment. Palm-Beclin 1 was detected by streptavidin pulldown followed by western blots. i, j, DHHC5-WT and DHHC5-KD (i) or clonal KO (j) H4 cells were subjected to IP-ABE assay with or without HAM treatment. Palm-Beclin 1 was detected by streptavidin pulldown followed by western blots. Similar results were observed in n = 3 independent experiments (c-e, g-j). Bars represent mean ± s.e.m. of n = 3 independent experiments (d-f). Bars represent mean ± s.d. of 3 independent samples (g). One-way ANOVA, post hoc Dunnett’s test (f).
