Western Blot Analysis of Cellular Proteins

Briefly, cells were lysed using MPER lysis buffer (Thermo #78501) supplemented with 5% protease (Sigma #P8340) and 1% phosphatase inhibitors (Phosphatase Inhibitor 2 -Sigma P5726 and Phosphatase Inhibitor 3—Sigma P0044). Between 25–50 μg of protein for each sample were separated by electrophoresis through an 8% SDS-polyacrylamide gel and transferred to a PVDF membrane (Immobilon). Blots were blocked in 5% milk, 1% bovine serum albumin (BSA), tris-buffered saline and tween 20 (TBST) for 1 hour at room temperature. Blots were incubated overnight at 4°C and probed with the following primary antibodies: MARCKS (1:1000, Abcam, Ab52616), actin (1:1000, Santa Cruz, sc-1616), V-5 (1:5000, Invitrogen, R96125), lamin A/C (1:1000, Santa Cruz, sc-6215), α-Tubulin (1:2000, Santa Cruz, sc-53646). After overnight incubation, blots were washed 3 x 5 min washes in TBST. Approximately 1:5000 dilution of the appropriate secondary antibodies in 5% milk, 1% BSA, TBST were used and blots were incubated for 1 hour at room temperature. Bands were detected via a chemiluminescence approach as described previously and following PerkinElmer’s instructions (Western Lightning Plus ECL, NEL102001EA) [1 (link)].