Rabbit anti p erk1 2

Western blot was carried out using the protocol described previously [31 (link)–33 (link)]. The following primary antibodies were used: rabbit anti-XPC (Santa Cruz, USA), rabbit anti-ERK2 (Santa Cruz, USA), rabbit anti-p-ERK1/2 (Santa Cruz, USA), rabbit anti-Snail (Santa Cruz, USA), anti-E-cadherin (Santa Cruz, USA) and rabbit anti-GAPDH (Santa Cruz, USA).

Rabbit anti-p-Akt (Ser473), anti-p-Akt (Thr308), anti-Akt, anti-PDK1, anti-p-PDK1, anti-PI3K p85, anti-p-PI3K p85, and anti-ERK1/2 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-CLDN2, rabbit anti-CLDN2, mouse anti-ZO-1, and rabbit anti-ZO-1 antibodies were from Thermo Fisher Scientific (Rockford, IL, USA). Mouse anti-p-Stat3 (Y705) and anti-Stat3 antibodies were from BD Biosciences (Franklin Lakes, NJ, USA). Goat anti-β-actin and rabbit anti-p-ERK1/2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fisetin, GEF, and LY-294002 were obtained from Cayman Chemical (Ann Arbor, MI, USA) and dissolved in dimethyl sulfoxide (DMSO). Control cells were treated with DMSO as a vehicle. The concentration of DMSO in the control and drug-treated cells was 0.1%. CDDP and DXR were from Fujifilm Wako Pure Chemical Industries (Osaka, Japan). DOC and hypoxia probe solution (LOX-1) were from Tokyo Chemical Industry (Tokyo, Japan) and Medical and Biological Laboratory (Tokyo, Japan), respectively. All other reagents were of the highest purity available.

The tissues or cells were homogenized in lysis buffer (50 mM Tris, pH 7.4, 150 mMNaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) containing protease inhibitors (Roche Applied Science). The lysates were clarified by centrifugation at 13,000 × g at 4 °C for 20 min, and the supernatants were collected and normalized for protein concentration. Proteins were separated by 8% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore). After blocking with PBS containing 5% skim milk and 0.05% Tween 20, the membranes were incubated with primary antibodies.For detection, a fluorescence-conjugated secondary antibody and an electrogenerated chemiluminescence system (GE Healthcare) were used. The membrane was exposed to an imaging system (LAS-3000, Fujifilm) according to the manufacturer’s specifications. The protein bands were quantified using ImageJ 1.44p software. The following antibodies were used: rabbit anti-p-ERK1/2 (1:1000,Santa Cruz, sc-7383); rabbit anti-ERK1/2 (1:1000,Santa Cruz,sc-292838), rabbit anti-GAPDH (1:5000, Abcam, ab128915). Horseradish peroxidase-conjugated rabbit IgG-specific (1:5000, Cell Signaling Technology, 7074 S) were used for secondary antibodies. Full-length gel is available at Supplementary data.