Hrp labeled secondary antibodies

Protein concentrations in cell and tumor lysates were quantified using the DC Protein Assay (Bio-Rad,). 20 μg of proteins were mixed with Laemmli buffer, boiled for 5 min, resolved by SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories). Membranes were blocked with 3% BSA for 1 hour and incubated with primary antibodies (4C, 16 hours). After 3 TBS/T washes, membranes were incubated with HRP-labeled secondary antibodies (Promega) for 1 hour at room temperature. Protein bands were developed using ECL Plus Detection Reagents (GE Healthcare). To analyze multiple proteins on the same membrane, membranes were washed with Restore PLUS Western Blot Stripping Buffer (Thermo Scientific) according to the manufacturer's protocol.

Protein concentrations in cell lysates were quantified using the DC Protein Assay (Bio-Rad). 20 μg of proteins were mixed with Laemmli buffer, boiled for 5 min, resolved by SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories). Membranes were blocked with 3% BSA for 1 hour and incubated with anti-mouse COX-2 polyclonal antibody (Cayman, Ann Arbor, Michigan), anti-mouse β-actin monoclonal antibody (Sigma, Saint Louis, Missouri), anti-mouse GAPDH monoclonal antibody (Santa Cruz Biotechnology, Inc, Dallas, Texas), anti-mouse ERK1/2 monoclonal antibody (Santa Cruz Biotechnology, Inc, Dallas, Texas) and anti-mouse phospho ERK1/2 monoclonal antibody (Cell Signaling, Danvers, Massachusetts) at 4°C for 16 hours. After 3 TBS/T washes, membranes were incubated with HRP-labeled secondary antibodies (Promega, Madison, Wisconsin) or IRDye Secondary Antibodies (Li-COR, Inc, Lincoln, Nebraska) for 1 hour at room temperature. Protein bands were developed using ECL Plus Detection Reagents (GE Healthcare) or Azure Biosystems C600 Imager.

Protein concentrations in cell and tumor lysates were quantified using the DC Protein Assay (Bio-Rad, Hercules, CA). 20 μg of proteins were mixed with Laemmli buffer, boiled for 5 min, resolved by SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked with 5% nonfat milk/PBS for 1 hr and incubated with primary antibodies (4°C, 16 hrs). After 3 TBS/Tween washes, membranes were incubated with HRP-labeled secondary antibodies (1:10,000 dilution, Promega, Madison, WI) for 1 hr at room temperature. Protein bands were developed using ECL Plus Detection Reagents (GE Healthcare, Piscataway, NJ) and quantified by densitometry with QuantityOne software (Bio-Rad Laboratories, Hercules, CA). To analyze multiple proteins on the same membrane, membranes were washed with Restore PLUS Western Blot Stripping Buffer (Thermo Scientific, Rockford, IL) according to manufacturer’s protocol.