Figure 5.

NRBF2 interacts and colocalizes with CCZ1-MON1A. (A) NRBF2 interacts with CCZ1 or MON1A. After N2a cells transiently transfected with Flag or Flag-Nrbf2 with GFP-Ccz1 or GFP-Mon1a, followed by immunoprecipitated (IP) with anti-Flag antibody; eluates were resolved by SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies. (B and C) Endogenous NRBF2 interacts with endogenous CCZ1 and MON1A in N2a cells as reflected by immunoprecipitation. (D) Endogenous CCZ1 interacts with endogenous NRBF2 and MON1A in N2a cells as reflected by immunoprecipitation. (E and F) Colocalization of NRBF2 with CCZ1 or MON1A. After HeLa cells transiently co-expressed NRBF2-HA with GFP-MON1A or GFP-CCZ1, the colocalization was visualized and representative images are shown. Scale bar: 7.5 μm. (G) Recombinant GST-NRBF2 protein directly binding with recombinant MON1A as reflected by GST affinity-isolation assay. (H) Endogenous CCZ1 interacts with NRBF2-CFP and dCCD-CFP. After N2a cells transiently expressed CFP, NRBF2-CFP or NRNF2-CFP mutant either missing CCD or MIT domain plasmids in nrbf2^-/- N2a cells, followed by immunoprecipitated (IP) with anti-CCZ1 antibody; eluates were resolved by SDS-PAGE and analyzed by immunoblotting with the corresponding antibodies. (I) Flag-MON1A interacts with NRBF2-CFP and dCCD-CFP in nrbf2^-/- N2a cells as reflected by immunoprecipitation. Irrelevant blots were removed. (J-L) The interaction between NRBF2 and CCZ1/RAB7 under normal and starvation conditions is determined by IP with an anti-NRBF2 antibody. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, vs. the relative control