-
A
U2OS cells were transiently co‐transfected with myc‐Atg4B and GFP‐Rab7b, incubated in CM or starved for 2 h, and lysed, followed by co‐IP with GFP‐Trap magnetic agarose beads. Whole‐cell lysates (WCL) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies.
-
B
U2OS cells were transiently co‐transfected with HA‐Rab7b Q67L and myc‐Atg4B, starved for 2 h, and lysed, prior to co‐IP with an antibody against myc or an isotype control (IgG1). Whole‐cell lysates (WCL) and immunoprecipitates (IP) were subjected to Western blot analysis using the indicated antibodies.
-
C
Coomassie blue staining of bacterially expressed His‐Rab9 Q66L, His‐Rab7b Q67L, and His‐Rab9 wt purified using cobalt‐coated Dynabeads.
-
D
Double immunogold labeling of U2OS cells stably transfected with GFP‐LC3 and transiently transfected with HA‐Rab7b. HA‐Rab7b (10 nm gold) localizes to outer (open arrowheads) and inner (filled arrowheads) membranes of autophagosomes, identified by LC3 labeling (5 nm gold, arrows) and characteristic ultrastructure (cytoplasmic mass surrounded with electron lucent area representing double membrane). Scale bars: 200 nm.
-
E, F
Live‐cell imaging of U2OS cells transiently transfected with mCherry‐LC3 and GFP‐Rab7b T22N (E) or mCherry‐LC3 and GFP‐Rab7b Q67L (F). Red and white arrows indicate vesicles positive for both mCherry‐LC3 and GFP‐Rab7b Q67L. Scale bar: 15 μm.
