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. 2014 Apr 14;289(22):15259–15271. doi: 10.1074/jbc.M114.559393

FIGURE 6.

FIGURE 6.

Hsp90-sGC interaction dynamics in response to heme-dependent versus heme-independent sGC activators. COS-7 cells expressing a V5-tagged heme-free mutant sGC-β1H105F or heme-deficient (SA-pretreated) RFL-6 cells expressing endogenous apo-sGC were given the heme-independent activator BAY 60-2770 (10 μm), and supernatants were made at 0, 15, 30, and 45 min. Parallel experiments utilized the heme-dependent activator BAY 41-2272 (10 μm). A and B, gel and Western analysis of immunoprecipitations with anti-V5 and sGC-β1 antibodies showing hsp90 associated with sGC-β1H105F or apo-sGC-β1, respectively (input 20%). C, cGMP concentrations in supernatants as indicated. D, hsp90 associated with sGC-β1 (input 20%) after cell treatment with BAY 41-2272. E, gel filtration fractions of supernatant from SA-pretreated RFL-6 cultures given BAY 60-2770 for 30 min, analyzed by Western blotting using sGC-β1 or hsp90 antibodies. Scale indicates Mr range of column fractions determined with protein Mr standards. Values in the bar graph are mean ± S.D. of three independent experiments. IB, immunoblot.