Gapdh

MPGs were excised and homogenized in a Tris-HCL (pH 7.5) buffer to evaluate the protein level of ROCK1 and ROCK2 by Western blot12 (link). Sol-O-Buffer (Fabgenix, Frisco, TX) was used to assay nNOS monomers and dimers in the MPG under non-reducing conditions38 (link). Protein (30 μg) was loaded on a 4–12% NuPage Bis-Tris gel (Invitrogen, Carlbad, CA) and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis was done at 4 °C for nNOS monomers and dimers. Proteins were transferred to polyvinylidene fluoride membranes and incubated with primary antibodies (ROCK1, ROCK2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), total casapse-3, activated caspase-3 (1:1000 Cell Signaling, Beverly, MA), nNOS (1:200 Santa Cruz), GAPDH (1:2500 BD Bioscience) and actin (1:500 (Sigma Aldrich, St. Louis, MO)) overnight at 4 °C. The membranes were incubated with a horseradish peroxidase-linked secondary antibody and visualized using an enhanced chemiluminescence kit (Amersham). The densitometry results were quantified using software Image J (National Institutes of Health) and normalized by actin level37 (link).

Cells were lysed in a 1% SDS (v/v) extraction buffer containing an anti-protease cocktail (Roche). Protein concentrations were determined using the Bradford assay (MicroBCA, Pierce). After resolution by SDS-PAGE, electrophoresed proteins were transferred to polyvinylidene fluoride (PVDF) membranes that were blocked and probed with the following antibodies: GRP94 (1:200, sc1794, Santa Cruz Biotechnology), HexA (1:100, sc-376777, Santa Cruz Biotechnology), GM2-AP (1:100, sc-514437, Santa Cruz Biotechnology), GAPDH (1:2500, BD Pharmingen) and the corresponding peroxidase-conjugated secondary antibodies at 1:2000. Immunoreactive bands were quantified using a VersaDoc™ (Bio-Rad) Imaging System using the Super Signal west-Pico (Pierce). Molecular weights were established with See Blue Plus2 Pre-stained Standard (Invitrogen).

Western blot analysis and cell fractionation were carried out as described previously [32 (link)]. Labelled bands were detected by Immun-Star horseradish peroxidase chemiluminescence kit (Bio-Rad, Regents Park, NSW, Australia), and images were captured with the Fujifilm LAS-4000 image system (Fujifilm, Brookvale, NSW, Australia). Antibodies included in this study were against PD-L1 (Rand D, Minneapolis, MN, USA) p-ERK, t-ERK, cyclin D1, STAT3, p50/105, RelA/p65 (D14E15), actin, tubulin, PARP (Cell Signaling, Danvers, MA, USA); XIAP (20/hILP/X, BD) GAPDH, c-Jun, p-c-Jun and vinculin (Santa Cruz technologies, Dallas, TX, USA).

The western blots were performed as previously described [35 (link)]. The antibodies used for western blots: anti- CD38 was from BD Biosciences (San Jose, CA), GAPDH, c-Raf, Phospho-c-Raf (S259), Mek, p-Mek, Erk, p-Erk, FAK, Lyn, Fgr, p47phox, Slp-76, Vav1, pY416-c-Src, horseradish peroxidase anti-mouse, and anti-rabbit antibodies, were from Cell Signaling (Danvers, MA), p-Tyr and c-Cbl antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), Phospho-c-Raf (S621) was from Thermo Fisher Scientific (Waltham, MA).

Antibodies against fibronectin, DCX, E2F3, TGFBR1 and WEE1were purchased from Santa Cruz Biotechnology (CA, USA). phospho-Akt, SIRT1, FBXW7, SNAIL and vimentin were purchased from Abcam (Cambridge, UK), and total Akt, N-cadherin, E-cadherin, NTRK3, RELN, EGFR, GAPDH and BMI1were from BD Biosciences (USA). HRP-conjugated goat anti-rabbit IgG was purchased from Santa Cruz Biotechnology. Total protein was extracted from the transfected cells and gastric cancer tissues using RIPA lysis buffer (Beyotime, China) according to the manufacturer's instructions. After the whole-cell protein extracts were quantified using a BCA protein assay, equivalent amounts of cell lysates were resolved by 10% SDS polyacrylamide gel electrophoresis and were transferred onto a polyvinylidene fluoride membrane, which was then blocked in 5% non-fat milk in TBST for 1 h at 4°C. The blots were then incubated with primary antibodies. After incubation with HRP-conjugated secondary antibodies, the protein bands were visualized using an enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA).