Figure 7
RNF182 physically interacts with ATP6V0C. A. Interaction between RNF182 and ATP6V0C in yeast two-hybrid system. Empty or RNF182 containing pGBKT7 bait vector and empty or ATP6V0C containing pACT2 library vector were co-transformed into yeast host cells AH109 and plated onto SD/-Trp -Leu -Ade -His +X-α-gal plate. In: a – a negative test of empty bait vector and ATP6V0C; b – a positive test showing the interaction between RNF182 and ATP6V0C; c – a negative test of RNF182 bait plus empty library vector. B. Total cellular proteins were extracted from HEK-293 cells co-transfected with flag-tagged ATP6V0C and GST-tagged RNF182 constructs and precipitated with glutathione-sepharose beads as described in the Materials and Methods. The precipitates were separated by 12% SDS-PAGE and transferred onto nitrocellulose membrane. The presence of RNF182 fragments in the complex was revealed by Western blotting with anti-GST antibody. Lanes 1, 3, 5, 7 represent total cellular proteins extracted from cells co-transfected with ATP6V0C and GST-RNF182 RING finger domain, GST-RNF182 C-end domain, GST-RNF182 full length, or GST alone, respectively. Lanes 2, 4, 6, 8 indicate GST fused protein fragments precipitated by glutathione-sepharose beads from cells co-transfected with ATP6V0C and GST-RNF182 RING finger domain, GST-RNF182 C-end domain, GST-RNF182 full length, or GST alone, respectively. C. The presence of ATP6V0C in the co-precipitated protein complexes shown in B (lanes 2, 4 6, 8) was revealed by Western blotting using anti-flag antibody. In: lane RINGΔ69–247 – GST-RNF182 RING finger domain, lane C-endΔ1–68 – GST-RNF182 C-end domain. D. Co-localization of RNF182 and ATP6V0C. N2a cells were co-transfected with flag tagged ATP6V0C and EGFP tagged RNF182 for 24 h. Cells were fixed and stained with anti-flag antibodies. Cy3-conjugated anti-rabbit IgG was used to detect the specific immunostaining. The nuclei were stained with DAPI and viewed with a Zeiss Axiovert 200 M × 40 fluorescence microscope. a. DAPI stained nuclei. b. EGFP tagged RNF182. c. Flag tagged ATP6V0C. d. a, b and c overlay.