Super signal femto reagent

Lysates of MCF-7 cells transfected with 1 µg of plasmid DNA or 50 nM siRNA were run on SDS–PAGE 6% polyacrylamide Tris-glycine gels and transferred to a polyvinylidene fluoride membrane. The membrane was blocked in phosphate-buffered saline containing 5% skim milk and 0.05% Tween-20 and incubated with antibody specific to each NM-II isoform or tubulin overnight at 4°C. The blots were washed and incubated with secondary antibody conjugated with horseradish peroxidase for 2 h at room temperature, followed by the addition of Super-Signal Femto reagent (Thermo Fisher Scientific). The luminescence signal was captured on Kodak x-ray film, and band intensity was quantified by ImageJ software (National Institutes of Health, Bethesda, MD).

Extracts of primary cells and tumor tissues were prepared in an extraction buffer composed of 50 mM Tris-HCl (pH 8.0), 60  mM KCl, 10 mM MgCl₂, 5 mM ATP, 4 mM EDTA, 1 mM dithiothreitol, 1% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, and protease inhibitors cocktail and RIPA buffer, respectively, at 4°C. Cell lysates were sedimented at 10,000 × g for 10 min. NMIIA and NMIIB were immunoprecipitated with antibody specific to NMHCIIA (Abcam) and NMHCIIB (Covance), respectively, using SureBeads Protein G Magnetic Beads (Bio-Rad, CA) as per the manufacturer’s protocol. Immunoglobulin G (IgG) was used as negative control for immunoprecipitation. Immunoprecipitates, cell or tumor tissue lysates were fractionated by SDS–PAGE on 8% or 10% polyacrylamide Tris-glycine gels, and transferred onto 0.45 µm polyvinylidene difluoride membranes (Millipore) as previously published (Saha et al., 2011 (link); Das et al., 2015 (link)). Membranes were blocked with 5% BSA or nonfat milk (Sigma-Aldrich) and incubated overnight at 4°C with primary antibodies specific to NMIIA, NMIIB, α-tubulin, β-actin (Sigma-Aldrich), Nesprin-2 (Abcam), or GAPDH (Santa Cruz). Membranes were then incubated with HRP-conjugated secondary antibody (Thermo Fisher) for 2 h. Chemiluminescence signal was visualized by Super Signal Femto Reagent (Thermo Fisher) and captured using a ChemiDoc Touch Imaging system (Bio-Rad).