Ibright cl750 system

Cells were lysed in 1X Laemmli buffer (65 mM Tris-HCl, pH7.0, 2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, 10% (v/v) glycerol, and 0.5% (w/v) bromophenol blue) and homogenized using a 28-gauge insulin syringe (BD). Equal protein masses of lysates were separated by electrophoresis in 7.5% PAGE gels, followed by transfer to PVDF membranes (Immobilon-P, Millipore). Membranes were incubated with primary antibodies overnight at 4 °C and the HRP-conjugated secondary antibodies at room temperature for 1 h. Blots were incubated with Super Signal West Pico (Thermo) and exposed to X-ray film or imaged using an iBright CL750 system (Thermo Fisher).

Cell pellets were lysed in RIPA buffer (Emd Millipore, 20–188) with complete protease (Sigma, 11836153001) and phosphatase (Sigma, 04906837001) inhibitor cocktails. Lysates were collected by centrifugation, 18,000 × g for 15 min at 4 °C. Protein concentration was measured using Bradford BCA colorimetric assay (Bio-Rad, 5000006). In all, 15 µg of sample lysates were subjected to western blot analysis using 10% Tris-Glycine gel under reducing conditions. Proteins were transferred onto PVDF membranes (Emd Millipore, IPVH00010) and probed with the following primary antibodies: COX-2 [74 kDa] at 1:1000 (Cell Signaling Technologies, 12282 S); GAPDH [~37 kDa] at 1:2000 (Santa Cruz biotechnology, SC-32233); and HMGB1 [~29 kDa] at 1:1000 (Biolegend, 651402). Secondary antibodies were purchased from the following sources: anti-mouse-HRP at 1:10,000 (Boster, BA1075) and anti-rabbit-HRP at 1:10,000 (Cell Signaling Technologies, 7074 S). Western blot bands were visualized using an enhanced chemiluminescence system (Thermo, 32106) on the iBright™ CL750 system and iBright Analysis Software version 1.5.0.