RIPA Lysis and Extraction Buffer

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	Cells were washed with ice-cold PBS and lysed in ice-cold radioimmunoprecipitation assay lysis buffer (Pierce), with added protease inhibitor (Sigma).

Protocol tips
Cells were washed with ice-cold PBS and lysed in ice-cold radioimmunoprecipitation assay lysis buffer (Pierce), with added protease inhibitor (Sigma).

Publication protocol

Cells were washed with ice-cold PBS and lysed in ice-cold radioimmunoprecipitation assay lysis buffer (Pierce), with added protease inhibitor (Sigma). In cell lysate, protein quantitation was done using BCA reagent (Pierce), as per manufacturer’s instructions. Thirty micrograms of protein sample was taken in fresh tubes and an equal volume of 2× Laemilli buffer was added to the cell lysate and boiled at 95°C for 10 min. The lysates were separated on SDS/PAGE (10% gel). The proteins on the gels were transferred oo PVDF membrane (Millipore). Then the blots were probed with an antibody specific to FRG1 (Novus Biologicals), and anti-mouse IgG secondary antibody (Pierce). The labeled bands were subsequently detected by chemiluminescence. For each sample, band intensities were normalized to GAPDH (Sigma) and β-tubulin (Cell Signaling Technology).

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