Immobilon psq polyvinylidene fluoride 0.2 μm pore size membranes

BM and spleen samples were homogenized in RIPA buffer containing 1X HALT protease inhibitor cocktail (78437; Thermo Scientific; Rockford, IL) and phosphatase inhibitor cocktail set IV (524628; Calbiochem, USA). Equal amounts of lysates were resolved by SDS-polyacrylamide gel electrophoresis (10% gels) and transferred to Immobilon-PSQ polyvinylidene fluoride 0.2μm pore size membranes (Millipore; Billerica, MA). Odyssey Blocking Buffer (LI-COR Biosciences; Lincoln, NE) was used to prevent non-specific binding. Immunoblotting was performed overnight at 4°C with diluted antibodies against Flag M2 (1:10,000; Sigma-Aldrich; St. Louis, MO), GFP (1:1000; Cell Signaling; Danvers, MA), VCAM-1 (1:1000; Santa Cruz Biotechnologies; Santa Cruz, CA), β-tubulin (1:1000; Cell Signaling), β-actin (1:1000; Santa Cruz) or GAPDH (1:1000; Cell Signaling). After washing with TBS-T, membranes were incubated at room temperature for 60 min with the appropriate diluted secondary antibody (IRDye680 Donkey anti-rabbit IgG (H + L) at 1:20,000; IRDye800CW Goat anti-mouse IgG (H + L) at 1:15,000; LI-COR Biosciences; IRDye680 Donkey anti-goat IgG (H+L) at 1:20,000). Bound antibody was detected using the LI-COR Biosciences Odyssey System (LI-COR Biosciences). Intensities were normalized to corresponding GAPDH, β-tubulin and β-actin intensities.