Acting on the CS domain of SHQ1, all components of the R2TP complex are required for SHQ1 removal from NAP57. (A) SHQ1 bound to MBP-NAP57 (lanes 1–3) or to control MBP-MCP (lanes 4,5) in amylose resin pull-down assays as in Figure 3C–E, except that the SDS-PAGE was transferred to nitrocellulose and stained with amido black (upper panel) and probed with pontin antibodies (lower panel). The bound proteins were incubated with S100 that was pretreated with (+) and without (−) pontin antibodies. (B–H) Same as A but coomassie blue stained gels. (B) SHQ1 bound to MBP-NAP57 (lane 1) was released with S100 extract (lane 2), which was pretreated with pontin antibodies (lane 3) that were incubated with 0.5, 1.5, and 2.5 μg of recombinant pontin (lanes 4–6) or reptin (lanes 7–9). (C) Same as B but with reptin antibodies. (D) MBP-NAP57 was incubated with the SHQ1 constructs indicated above the gel and treated with (+) and without (−) S100. Note the SSD bound to MBP-NAP57 was not removed by S100 (lanes 3,4). (E) The CS domain of SHQ1 bound in trans to its SSD to MBP-NAP57 was removed by S100 (lane 2), which was inhibited by pontin and reptin antibodies (lanes 3,4). (F) PIH1D1 antibodies inhibited S100-mediated removal of SHQ1 from MBP-NAP57 (lane 3). The inhibition was relieved by recombinant PIH1D1 (lane 4). (G) Same as F but with RPAP3 antibodies and protein. (H) Control. Unlike pontin antibodies (lane 6), antibodies directed against NAP57, SHQ1, and NAF1 did not inhibit the release activity of S100 extracts (lanes 3–5, respectively).
