Western blotting and immunoprecipitation were performed as previously described [32 (link)]. Briefly, cell lysates were prepared using lysis buffer containing Tris pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 50 mM NaF, 10 mM β-glycerophosphate, 10 nM calyculin A, 1 mM Na3VO4 and protease inhibitors, and normalized by protein concentrations using the Bradford method (Bio-Rad). For western blotting, cell lysates were boiled in Laemmli sample buffer and separated on 8%–12% SDS-PAGE and transferred to Immobilon-P PVDF Membrane (Sigma-Aldrich). The membranes were blocked in TBST containing 5% nonfat milk, incubated with primary antibodies based on the manufacturer’s instructions, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Thermo Fisher) and enhanced chemiluminescence detection (Sigma-Aldrich).
For co-immunoprecipitation, cell lysates were incubated with primary antibodies, anti-FLAG M2 affinity agarose gel, or anit-HSP90 conjugated agarose at 4 °C overnight, followed by incubation with protein A/G Sepharose for an additional 1 h at 4 °C, when applicable. Beads were washed three times with lysis buffer and boiled in Laemmli sample buffer, and immune complexes were analyzed by SDS-PAGE and western blotting.
For co-immunoprecipitation, cell lysates were incubated with primary antibodies, anti-FLAG M2 affinity agarose gel, or anit-HSP90 conjugated agarose at 4 °C overnight, followed by incubation with protein A/G Sepharose for an additional 1 h at 4 °C, when applicable. Beads were washed three times with lysis buffer and boiled in Laemmli sample buffer, and immune complexes were analyzed by SDS-PAGE and western blotting.
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