Criterion tgx 4 20 gels

Cells were washed in PBS and lysed in RIPA buffer (St. Louis, MO, USA cat# R0278) supplemented with 10 % phosphatase (Thermo Fisher cat# 1862495) and protease inhibitors (Thermo Fisher cat# 186178). Lysates were then clarified by spinning at 14,000 rpm at 4°C and protein concentration quantified by Bradford assay. 50 μg of protein was boiled in loading buffer for three minutes, electrophoresed in Criterion TGX 4–20% gels (Bio Rad, Hercules, CA, USA cat# 5671095), and transferred to nitrocellulose membranes. Blots were blocked in 5% milk/TBST (20mM Tris pH7.5, 150 mM NaCl, 0.5% Tween 20) solution and stained at 4°C overnight with primary antibodies diluted in 2% BSA (bovine serum albumin)/TBST. The antibodies used were anti-JAM-1 (1:1000; cat#Ab52647 Abcam Cambridge, MA), GAPDH (1:1000; cat#Ab9485 Abcam Cambridge, MA), acetylated histone 3 lysine 27 (Ac-H3-K27; 1:1000; cat#Ab729 Abcam Cambridge, MA), and anti-RV σ-NS (1:1000; provided by Oncolytics Biotech Inc.). Blots were washed three times for 15 minutes with TBST, and stained with horseradish peroxidase (HRP)-conjugated secondary antibodies (diluted 1:4000) for 2 hrs at room temperature. Following two 10-minute washes in TBST, signals were detected with Super Signal HRP substrate (Thermo Fisher cat# 34080).