To fractionate C2C12 cells into cytosolic and membrane fractions, we first washed a 10‐cm dish with cold PBS and lysed the cells by homogenization in hypotonic buffer (10 mM Tris pH 8.0, 1 mM ethylenediaminetetraacetic acid [EDTA]) supplemented with 1 mM phenylmethane sulfonyl fluoride (PMSF) and a protease inhibitor mix. The homogenate was centrifuged at 15,000 revolutions per minute (RPM) for 5 min to pellet nuclei and cell debris. The supernatant was centrifuged at 12,000 RPM for 20 min to pellet membrane structures. The supernatant from this step was the cytosolic fraction, and the membrane fraction was solubilized in an equal volume of hypotonic buffer + 1% n‐dodecyl β‐d ‐maltoside (DDM; Sigma‐Aldrich, St. Louis, United States) for further analyses by immunoblotting.
For analysis of whole‐cell extracts, Radioimmunoprecipitation assay buffer (RIPA buffer) (10 mM Tris–Cl [pH 8.0], 1 mM EDTA, 0.5 mM egtazic acid (EGTA), 1% Triton X‐100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 140 mM NaCl) supplemented with 1 mM PMSF and a protease inhibitor mix was used for cell lysate preparation.
For immunoblotting, equal protein amounts were separated by 12% SDS–polyacrylamide gel electrophoresis, transferred to a PVDF membrane (Millipore, MA), blocked in 5% milk in TBS‐tween, and incubated with primary antibodies.
For analysis of whole‐cell extracts, Radioimmunoprecipitation assay buffer (RIPA buffer) (10 mM Tris–Cl [pH 8.0], 1 mM EDTA, 0.5 mM egtazic acid (EGTA), 1% Triton X‐100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 140 mM NaCl) supplemented with 1 mM PMSF and a protease inhibitor mix was used for cell lysate preparation.
For immunoblotting, equal protein amounts were separated by 12% SDS–polyacrylamide gel electrophoresis, transferred to a PVDF membrane (Millipore, MA), blocked in 5% milk in TBS‐tween, and incubated with primary antibodies.