β actin antibody

Total RNA was extracted using the TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. Complementary DNA was synthesized from 2 μg of total RNA using random hexamers (Proligo, Boulder, CO) and SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA). RT-PCR was carried out on a panel of cell lines and tumor samples. The real-time quantitative reverse transcription PCR (qRT-PCR) conditions were as follows: 1 min at 95 °C, denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, and extension at 72 °C for 60 s. As follows are the primers of USP7: 5′-GTTGTTGGAGCGATTACAAGA-3′ and 5′-AAACTGGTCCTCTGCGACTATC-3′; β-actin: 5′-GGCATCCTGGGCTACACTGA-3′ and 5′-GTGGTCGTTGAGGGCAATG-3′. Relative expression among samples was calculated by the comparative CT method. All experiments were performed in triplicate.
Thirty micrograms of total extract protein was run on SDS-polyacrylamide gel electrophoresis gels, transferred onto polyvinylidene difluoride membranes, and incubated with the corresponding antibodies. The membranes were developed with the enhanced chemiluminescence method (Pierce, Rockford, IL, USA). The primary antibodies used were USP7 (1:1,000, Abcam, UK). β-Actin protein detection using β-actin antibodies (1:2,000, Beyotime, China) was used as an internal control. All experiments were performed in triplicate.

Radioimmunoprecipitation (RIPA) with phenylmethanesulfonyl fluoride (PMSF) buffer and bicinchoninic acid (BCA) protein kit were ordered from Solarbio Life Science, China. Trifluoroacetic acid (TCA) was attained from Shanghai Macklin Biochemical Co., Ltd., China. The following primary antibodies were used for the Western bot of colon tissue: phosphorylated p38 (p-p38) and phosphorylated NF-κB (p-p65) antibodies (Cell Signaling, Danvers, MA, USA), Inhibitor kappa B-α (IκB-α), NF-κB (p65), p38, Claudin-1, Caspase-8/9/3, Bax, proliferating cell nuclear antigen (PCNA), and β-actin antibodies (Beyotime Institute of Biotechnology, Shanghai, China).