Ecl prime western blot substrate

The primary antibodies mouse anti-human AEG-1 antibody (sc-517220), mouse anti-human phosphatase and tensin homolog (PTEN) antibody (sc-7974), mouse anti-human AKT antibody (sc-56878), mouse anti-human p-AKT antibody (sc-271966), and mouse anti-human GAPDH antibody (sc-81545) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protein was isolated from tissue samples or cells using ice-cold lysis buffer (Beyotime Biotechnology, Jiangsu, P.R. China). A BCA Protein Assay Kit (Pierce; Thermo Fisher Scientific, Inc.) was applied to examine the concentration of total protein. Equal amounts of protein were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the fractionated proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Boston, MA, USA). After blocking with TBS/0.1% Tween (TBST) containing 5% skimmed milk at room temperature for 2 h, the membranes were incubated with the primary antibodies at 4°C overnight. The membranes were washed three times with TBST and then probed with goat anti-mouse horseradish peroxidase-conjugated secondary antibody for 2 h (Santa Cruz Biotechnology) at the dilution ratio of 1:5,000. The protein signals were visualized using an ECL prime Western blot substrate (GE, Amersham, UK). Relative protein expression was presented as the density ratio versus GAPDH.

Total protein was extracted from the tissues and cells using RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, P.R. China). A BCA assay kit was used to detect the total protein concentration. A specific amount of total protein was electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel and transferred into polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk for 1 h, the membranes were incubated with the primary antibodies overnight at 4°C, washed with Tris-based saline-Tween 20 three times, and incubated with corresponding horseradish peroxidase-conjugated secondary antibody (sc-2005; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Enhanced chemiluminescence (ECL) prime Western blot substrate (GE, Amersham, UK) was used to visualize the protein bands. The primary antibodies used in this study were acquired from Santa Cruz Biotechnology and included mouse anti-human HDGF antibody (sc-271344), mouse anti-human phosphotidylinositide 3 kinase (PI3K) antibody (sc-376412), mouse anti-human phosphorylated (p)-PI3K antibody (sc-56938), mouse anti-human p-AKT antibody (sc-271966), mouse anti-human AKT antibody (sc-81434), and mouse anti-human β-actin antibody (sc-69879). The densities of the protein bands were quantified using ImageJ 1.49 (National Institutes of Health, Bethesda, MD, USA).