Figure 7From: A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradationRNF182 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.Back to article page