SHQ1 can be released from NAP57 by cytosolic S100 extracts in an ATP- and HSP90-independent fashion. (A,C–E) Amylose resin pull-down assays as in Figure 1G–K. (A) SHQ1 bound to MBP-NAP57 (lane 2) was incubated with recombinant pontin, reptin, or both (2.5 μg each) in the presence (+) and absence (−) of 1 mM ATP. (B) Western blot of recombinant pontin (lane 1) and cytosolic S100 extract (lane 2) probed with pontin antibodies and developed with enhanced chemiluminescence (ECL). (C) SHQ1 bound to MBP-NAP57 (lane 1) was incubated with 1 μL and 10 μL of S100 extract in the presence (+) and absence (−) of 1 mM ATP. (D) SHQ1 bound to MBP-NAP57 was incubated with S100 that was pretreated with (+) and without (−) Apyrase (140 mU/μL) for 30 min at 37°C. (E) SHQ1 bound to MBP-NAP57 was incubated with S100 that was pretreated with the HSP90 inhibitor geldanamycin (GA) at 0, 4, and 8 μM for 60 min at 37°C.
