Horseradish peroxidase hrp conjugated antigoat igg

HT29 and MC38 colon cancer cell lines were grown in McCoy’s medium and DMEM containing 10% heat-inactivated fetal bovine serum (FBS) at 37 °C in a humidified 5% CO₂ incubator. Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) (MTS) assay kit (Promega), as described in the manufacturer’s protocol. Lithium chloride (LiCl), PD98509 and okadaic acid were purchased from Sigma (St. Louis, MO, USA). Antibodies for Western blot analysis were obtained from Cell Signaling Technology: anti-GSK-3β, anti-phosphorylated GSK-3β (anti-p-GSK-3β), anti-AKT, anti-phosphorylated AKT (anti-p-AKT), anti-β-catenin, anti-phosphorylated β-catenin (anti-p-β-catenin), anti-phosphorylated ERK (anti-p-ERK) and anti-ERK. Antibodies against Wnt, p53 and p21 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-TREM2 and TREM2-Ig were obtained from R&D Systems (Abingdon, UK). Anti-EpCAM and anti-a-tubulin antibodies were purchased from Abcam (Cambridge, UK) and Sigma, respectively. Horseradish peroxidase (HRP)-conjugated anti-goat IgG and anti-rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) were used as secondary antibodies for Western blot analysis.

Phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibiotics (amphotericin B, penicillin, and streptomycin) were purchased from Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), 2′,7′-dichlorofluorescein diacetate (DCF-DA), diphenylene iodonium (DPI), and other chemicals were obtained from Sigma (St. Louis, MI, USA). Antibodies were obtained as following sources: anti-phospho-AMPK pAb (sc-33524), anti-AMPK pAb (sc-25729), and anti-NOX4 pAb (sc-30141), anti-NOX2 (gp91phox, sc-5827) pAb, anti-Col I pAb (sc-25974) and anti-fibronectin pAb (sc-9068), and horseradish peroxidase (HRP)-conjugated anti-goat IgG) were purchased from Santa Cruz Biotechnology (SantaCruz, CA, USA) and anti-mouse IgG (#7076), and anti-rabbit IgG (#7074)were purchased from Cell Signaling Technology (Dancers, MA, USA).