Bradford bca colorimetric assay

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.

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 mg 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, 12282S); 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, 7074 S). Western blot bands were visualized using enhanced chemiluminescence system (Thermo, 32106) on autoradiography films (Genesee, 30-507) or the iBright™ CL750 systems. Western blot was quantified using the ImageJ software (ver. 1.5i) or the iBright analysis software (ver. 1.5. 0).