Protein g sepharose

After 48 h of transfection with the appropriate plasmids, the cells were collected and lysed in a lysis buffer (10% glycerin (w/v), 1 mmol/L EDTA, 50 mmol/L pH 7.5 Tris-HCl, 0.5% Triton X-100 (v/v), 150 mmol/L NaCl, 1 mmol/L PMSF). The products were interacted with Flag M2 affinity gel (Sigma–Aldrich) at 4 °C for 4 h. To explore the endogenous interaction proteins in MCF-7 or LM-MCF-7 cells, protein G-Sepharose (Santa Cruz Biotechnology) was incubated with the negative control IgG, or anti-NMHC-IIA, anti-PKCβII and anti-HBXIP, then were incubated with the cell lysate. After washing with the washing buffer for eight times, the affiliative proteins were washed out from the gel with washing buffer and then separated by SDS-PAGE and immunoblotting.

Reciprocal co‐immunoprecipitation (co‐IP) was performed as previously reported.35 In brief, 300 μg of solubilized proteins isolated from the adult mouse heart or from HEK293 cells was incubated with anti‐JP2, anti‐SK2 or anti‐RyR2 antibodies overnight at 4°C, and control groups (Ctrl‐N) were incubated with IgG antibodies of non‐mouse and non‐rabbit sources, followed by an additional incubation with protein G Sepharose (Santa Cruz Biotechnology, La Jolla, CA, USA) for 6 hour at 4°C. Protein‐bead complexes were washed three times with a high‐salt buffer (50 mmol L⁻¹ Tris pH 8.0, 1 mol L⁻¹ NaF, 500 mmol L⁻¹ NaCl, 0.5% NP‐40 and complete protease inhibitor mixture), then a low‐salt buffer (50 mmol L⁻¹ Tris pH 8.0, 1 mol L⁻¹ NaF, 250 mmol L⁻¹ NaCl, 0.5% NP‐40, and complete protease inhibitor mixture) and finally a salt‐free buffer (50 mmol L⁻¹ Tris pH 8.0, 1 mol L⁻¹ NaF, 0.5% NP‐40, and complete protease inhibitor mixture) to remove non‐specific binding. The immune complexes were assessed with immunoblot analysis with anti‐JP2, anti‐SK2 or anti‐RyR2 antibodies.

Cells were lysed 36–48 h after transfection of expression plasmids using 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100 containing cocktail. For immunoprecipitation, lysates were incubated with the appropriate antibodies for 2 h on ice, followed by precipitation with protein G Sepharose (Santa Cruz). Samples were separated by SDS–PAGE and transferred to PVDF membranes. After blocking in PBS containing 0.1% Tween-20 and 5% skim-milk, the blots were probed with indicated antibodies. Westernblot visualization was done with enhanced chemiluminescence.