Figure 2

PNRC associates with RPC39 in mammalian cells. MCF-7/EGFP or MCF7/EGFP-PNRC stable expression cells were transiently transfected with the RPC39 expression plasmid, pSG5-RPC39, 24 h post-transfection, cells were harvasted and lysed, and 15 mg of total proteins were immunoprecipitated with antibodies against GFP (Clontech) or RPC39 (Santa Cruz Biotechnology). Specifically bound proteins to Protein A agarose beads were separated on a 10% SDS-PAGE and analysed by immunoblotting with either anti-RPC39 (A, blot: anti-RPC39) or anti-GFP (B, blot: anti-GFP) antibody, as previously described [2]. An aliquot of cell lysate equal to 100 μg of protein was included in each SDS-PAGE gel for Western blot to examine whether the crude protein extracts used for co-immunoprecipitation contains EGFP-PNRC and RPC39 proteins. The protein bands with molecular weights corresponding to those of RPC39 or EGFP-PNRC were indicated by arrows.