Precision plus protein standards kaleidoscope

Whole cell lysate was prepared by solubilizing cell pellet in RIPA buffer (Sigma; R-0278) over ice for 40 minutes as described previously [32 (link)]. The lysate was cleared by centrifugation at 14000 g for 15 min at 4°C and the supernatant was collected as whole cell lysate. Cytosolic and nuclear fractions were prepared as described previously with minor modifications [33 (link)]. Protein estimation was done by Bradford’s method.
Western blotting was performed using equal amounts of protein, including pre-stained molecular weight markers (Precision Plus Protein Standards Kaleidoscope, Bio Rad). The proteins were electrotransferred to polyvinylidene difluoride (PVDF) membranes (Immobilon P 0.45 μm, Millipore). After blocking for 2 h at room temperature in 5% fat-free milk powder or 3% BSA in TBST (Tris-buffered saline containing 0.05% Tween 20, pH 8.0), membranes were incubated overnight with specific antibodies. After 30 minutes washing with TBST, membranes were probed with different HRP-conjugated secondary antibodies. Detection of specific proteins was carried out by Immobilon Western (Chemiluminescent HRP substrate, Millipore) mediated chemiluminescence by ChemiDoc XRS+ (Bio Rad). Densitometry of specific bands was done using Image Lab Software version 3 (Bio Rad).

Total protein content of the samples was determined using the potassium dodecyl sulphate method [24 (link)] and protein quantification was carried out using the bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich, Saint Louis, MO, USA) [18 (link)]. Electrophoretic separation (SDS-PAGE) was performed using a vertical electrophoresis apparatus (Mini-Protean Tetra cell, Bio-Rad, Richmond, CA, USA) and standard molecular weight (Precision Plus Protein Standards, Kaleidoscope, Bio-Rad, Richmond, CA, USA) [18 (link)].