Fig. 2: Atg7 degrades PDCD4 through the ubiquitin-proteasomal pathway.
a PDCD44 half-life is extended in HEK293 Atg7-KO cells compared with that in Atg7-WT cells. The cells were switched to fresh medium (10% FBS) containing 50 mg/mL cycloheximide (CHX) for the indicated time and harvested for Western blot assay. b PDCD44 half-life is shortened in the HEK293 cells that were transfected with Flag-Atg7 compared to those transfected with the control vector. The cells were switched to fresh medium (10% FBS) containing 50 mg/mL cycloheximide (CHX) for the indicated time and harvested for Western blot assay. c Western blot detection of PDCD4 in HEK293 Atg7-KO cells andAtg7-WT cells after incubating with 10 mmol/L MG132 for the indicated times. d Western blot detection of PDCD4 in HEK293 cells that were transfected with Flag-Atg7 or control vector incubating with 10 mmol/L MG132 for the indicated times. e Western blot detection of p-PDCD4 in HEK293 cells that were transfected with Flag-Atg7 or control vector incubating with Bafilomycin A1 (20 nM) for 12 h, then treated or not with 2DG (5 mM) and oligomycin (2.5 μM) stimulation for four hours. f In vivo ubiquitination assay in HEK293 Atg7-KO cells or Atg7-WT cells. g In vivo ubiquitination assay of HEK293 cells transfected with GFP-Atg7 or GFP-Atg7C571S and the indicated plasmids. h In vivo ubiquitination assay of HEK293 cells transfected with GFP-Atg7 after 2DG (5 mM) and oligomycin (2.5 μM) treatment for four hours.
