Figure 4
PMR60 associates with KSRP and with the exosome component Rrp4. A-B. KSRP-PMR60 interaction. A. HEK293 cells were transfected with plasmids for TAP and Myc-tagged PMR60 or TAP-tagged GFP without (middle panels, for endogenous KSRP) or with Flag-KSRP1-4 (bottom panels, for over-expressed Flag-KSRP1-4). TAP-PMR60 and TAP-GFP were affinity-purified followed by Tev protease cleavage and analyzed by immunoblots (IB) with the indicated antibodies, along side samples of the input (10% of the IPed fraction). The decreased size of the recovered PMR60 and GFP (top panel, bound) results from Tev protease cleavage of the TAP tags. B. HEK293 cells were co-transfected with expression plasmids for TAP-PMR60 as above and either Flag-KSRP or Flag-KSRP deletion mutants (Flag-KSRP1-4 and Flag-KSRP3-4). Cell extracts were divided into untreated (-) or RNase A and T1 treated (+) fractions. Input (10% of IPed fraction) and TAP-PMR60 purified fractions were analyzed by IB with anti-Flag antibodies. C. PMR600 co-immunoprecipitates with Rrp4. Cos-1 cells were co-transfected with plasmids expressing TAP- and Myc-PMR600 and either Flag-GFP or Flag-Rrp4. Cytoplasmic extracts were immunoprecipitated with anti-Flag agarose beads and analyzed by IB with the indicated antibodies. The dot in the upper blot indicates IgG heavy chain.