Bolt 4 12 bis tris plus precast gels

Micropellets were first washed with PBS, then stored in RIPA lysis buffer (Sigma Aldrich) containing a protease inhibitor cocktail (Sigma, P-8340) for 1 h at −80 °C. Micropellets were then grinded and centrifuged at 12,000 rcf, at 4 °C for 15 min. Total proteins (50 µg) diluted in Bolt LDS sample buffer and Bolt Sample Reducing Agent were heated at 70 °C for 10 min and loaded on Bolt 4–12% Bis–Tris Plus precast gels (Life Technologies). Samples were allowed to migrate for 30 min at 200 mV using the MOPS SDS running buffer and transferred on nitrocellulose membranes using the iBlot 2® Gel Transfer Device (Life Technologies). Membranes were then blocked with 5% milk and probed with the anti-human NMB rabbit polyclonal primary antibodies (1:500; Cohesion Biosciences, Nanterre) at 4 °C, overnight. Anti-human Actin mouse polyclonal primary antibodies (1:5000) for 2 h at room temperature. After several rinses with tris phosphate buffered saline (TBS) containing 0.05% tween-20 (Sigma-Aldrich), membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:50000) for 1 h at room temperature. Blots were revealed using a WesternBright™ Sirius Chemiluminescent detection kit (Advansta, Blagnac) and scanned (ChemiDoc XRS System, Biorad).

Prior to western blot analysis, cells were washed once with ice-cold phosphate buffered saline and lysed in lysis buffer (50 mM HEPES (pH 7.5), 150 mM KCl, 5 mM EDTA, 0.05% IGEPAL CA-630 (v/v), 1x protease inhibitor cocktail and 1x phosphatase inhibitor cocktail). Lysates were briefly sonicated before low speed centrifugation (900 g, 10 min at 4°C) with total protein concentration determined by Bicinchoninic Acid (BCA) protein assay. Equal amounts of lysate were denatured in 1x Laemmli buffer containing 8% β-mercaptoethanol by heating for 5 min at 80°C. Samples were subjected to SDS-PAGE analysis using Bolt 4%–12% Bis-Tris Plus pre-cast gels (Life Technologies) before protein transfer to nitrocellulose membrane (GE Healthcare Life Sciences) using the Novex system (Life Technologies). Membranes were blocked in Odyssey blocking buffer (Li-Cor Biosciences) and incubated with primary antibodies overnight at 4°C with gentle agitation. All antibodies were used at a 1:1,000 dilution with the exception of the anti-α-tubulin antibody (1:5,000). Membranes were washed with PBS containing 0.1% Tween-20, incubated with appropriate secondary antibodies and scanned using an Odyssey CLx imaging system (Li-Cor Biosciences). Images were subjected to densitometric analysis using ImageJ software.