Extended Data Fig. 3: Increased autophagy, but not endocytosis, in beclin 1(F121A) MEFs.
From: Disruption of the beclin 1–BCL2 autophagy regulatory complex promotes longevity in mice
a, Co-immunoprecipitation of beclin 1 and BCL2 in MEFs derived from Becn1+/+ (wild-type) and Becn1F121A/F121A (knock-in) animals. b, Representative images (top) and quantification (bottom) of GFP–LC3 puncta in wild-type and knock-in cells with or without 10 nM bafilomycin A1 (BafA1) for 3 h. Scale bars, 10 µm. c, Western blot analysis of autophagy markers in wild-type and knock-in MEFs with or without BafA1 (100 nM, 2 h). d, Representative images and quantitative electron microscopic analysis of autophagic structures in wild-type and knock-in MEFs with or without BafA1 (100 nM, 3 h). Insets show representative autophagosome (arrowhead) and autolysosome (arrow). Scale bars, 1 µm. e, Representative images and quantification of transferrin uptake kinetics in wild-type and knock-in cells. Scale bars, 20 µm. Results shown are representative of two and four independent experiments respectively for a and c. Data are mean ± s.e.m. for three replicates in b and for 50 cells per genotype and condition in d. Data points on line graph in e denote mean ± s.e.m. for cells at 7 min (n = 63, WT; n = 54, KI), 15 min (n = 58, WT; n = 52 KI), and 30 min (n = 76, WT; n = 83, KI). P values determined by unpaired one-sided (b) and two-sided (d, e) t-test. For uncropped gels, see Supplementary Fig. 1.
