Figure 4.
NRBF2 is required for RAB7 activation to promote autophagosome maturation. (A and B) GTP-RAB7 in WT and nrbf2-/- N2a cells was determined by GTP-beads affinity-isolation assay. The “empty agarose” indicated affinity isolation with empty agarose beads, the “pull-down” indicated affinity isolation with GTP-beads. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, vs. the relative control. (C and D) GTP-RAB7 in the WT and nrbf2-/- mice brain was determined by GTP-beads affinity-isolation assay. The “empty agarose” indicated affinity isolation with empty agarose beads, and the “pull-down” indicated affinity isolation with GTP-beads. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, vs. the relative control. (E and F) GST-R7BD affinity-isolation assay indicate that active RAB7 is reduced in nrbf2-/- cell. These findings are consistent with GTP-beads affinity-isolation assay. The “GST” indicated affinity isolation with GST only beads, and the “GST-R7BD” indicated affinity isolation with GST-R7BD beads. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, vs. the relative control. (G and H) GST-R7BD affinity-isolation assay indicates that active RAB7 is reduced in nrbf2-/-mouse. The GST means affinity isolation with GST-only beads, GST-R7BD means affinity isolation with GST-R7BD beads. These findings are consistent with GTP-beads affinity-isolation assay. (I and J) Rescue of decreased GTP-RAB7 in nrbf2-/- cells by overexpressing Flag-NRBF2. (K and L) Transiently overexpression of RAB7Q67L and RAB7 WT but not RAB7T22N promotes the degradation of SQSTM1 in nrbf2-/- cells. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, vs. the relative control. (M) Colocalization of GFP-LC3 and RFP-RAB7 in WT and nrbf2-/- N2a cells as seen under confocal microscope. Scale bar: 5 μm. (N) Colocalization of GFP-LC3, RFP-RAB7 and NRBF2 in N2a cells as seen under confocal microscope. Scale bar: 2.5 μm. (O) Transient overexpression of NRBF2 increases RAB7 activity as reflected by an assay using the RAB7 FRET sensor, Raichu-RAB7. The CFP/YFP ratio was obtained from microplate reader (Molecular devices FlexStation 3). (P) nrbf2 KO decreases RAB7 activity as reflected by an assay using the RAB7 FRET sensor, Raichu-RAB7. The CFP/YFP ratio was obtained from microplate reader (Molecular devices FlexStation 3). Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, vs. the relative control