Anti rabbit igg horseradish peroxidase conjugate

Western blotting assays were conducted using a previously published protocol [57 (link)]. Total proteins were extracted from CRC samples with RIPA buffer (Thermo Fisher Scientific, USA). The protein concentrations were measured with a BCA assay kit (Thermo Fisher Scientific, USA). An equivalent amount of protein from each sample was separated using SDS‒PAGE. Then, the proteins were transferred to polyvinylidene membranes (Millipore; Burlington, MA, USA). The membranes were blocked with Tris-buffered saline-0.1% Tween-20 (TTBS) supplemented with 5% skim milk for 2 h at 25 °C. The membranes were then incubated at 4 °C overnight with the following primary antibodies at the indicated dilutions: anti-CYP2W1 (1:500, PA5-101315, Invitrogen), anti-GDE1 (1:1000, PA5-43012, Invitrogen), anti-PTPN6 (1:1000, ab124942, Abcam), anti-PTTG1IP (1:500, ab128040, Abcam), anti-SEC61G (1:500, PA5-21384, Invitrogen), and anti-TRIP6 (1:500, ab137478, Abcam) antibodies. Next, the membranes were incubated with a secondary antibody, anti-rabbit IgG-horseradish peroxidase conjugate (1:4000; Cell Signaling Technology) for 1 h at 25 °C. The expression levels of the target proteins were detected using an ECL kit (Bio-Rad, USA) and analyzed via ImageJ software ((NIH V1.8.0.112, USA).

Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific. Sodium dodecyl sulfate-polyacrylamide electrophoresis apparatus, immunoblotting reagents were obtained from Bio-Rad Laboratories (Hercules, CA). Pierce MemCode Reversible Protein Stain Kit, BCA Protein Assay Kit and Pierce Detergent Compatible Bradford Assay Kit were from Thermo Fisher (Waltham, MA). Anti-phospho-AKT Ser473 (pAKTSer473; #9271), anti-phospho-AKT Thr308 (pAktThr308; #9275), anti-phospho-AKT2 Ser474 (pAKT2Ser474; #8599), anti-phospho-AS160 Thr642 (pAS160Thr642; #8881), anti-AKT1 (# 2938), anti-AKT2 (#3063), anti-Na⁺, K⁺ ATPase (#3010), anti-LDH (#3558), anti-insulin receptor (IR; #3025) and anti-rabbit IgG horseradish peroxidase conjugate (#7074) were from Cell Signaling Technology (Danvers, MA). Anti-AKT substrate of 160 kDa (AS160; ABS54) was obtained from EMD Millipore. Human recombinant insulin was from Eli Lilly (Indianapolis, IN).