a, Co-immunoprecipitation of beclin 1 and Bcl-2 in MEFs derived from Becn1WT/WT (WT) and Becn1F121A/F121A (KI) animals. b, Representative images (top) and quantitation (bottom) of GFP-LC3 puncta in WT and KI cells +/− bafilomycin A1 (BafA1) (10 nM, 3 h). Scale bars, 10 μm c, Western blot analysis of autophagy markers in WT and KI MEFs +/− Baf A1 (100 nM, 2 h). d, Representative images and quantitative electron microscopic analysis of autophagic structures in WT and KI MEFs +/− Baf A1 (100 nM, 3 h). Insets show representative autophagosome (arrowhead) and autolysosome (arrow). Scale bars, 1 μm. e, Representative images and quantitation of transferrin uptake kinetics in WT and KI cells. Scale bars, 20 μm. Results shown are representative of 2 and 4 independent experiments respectively for (a) and (c). Bars represent mean ± s.e.m for 3 replicates in (b) and for 50 cells per genotype and condition in (d). Data points on line graph in (e) represent 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 for indicated comparisons, unpaired t-test one-sided for (b) and two-sided for (d) and (e). WCL, whole cell lysate. For uncropped gels, see .