Anti tpm4

After the cells were digested with protein lysates, the total proteins of the AGS and BGC-823 cells were extracted. A BCA kit (Beyotime, P0010) was used for measuring the cellular protein content. 10% SDS-PAGE was used to separate different proteins, and 50 g of protein was loaded per lane. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes and blocked with 5% milk. The primary rabbit antibodies used were anti-TPM4 (cat number: ab181085; 1:1,000; Abcam) and anti-GAPDH (cat number: sc-32233; 1:1,000; Santa Cruz). A processing film containing HRP-conjugated goat anti-rabbit IgG (cat. no. sc-2004; 1,000; Santa Cruz) and anti-mouse IgG (cat. no. sc-2005; 1,000; Santa Cruz) was used. The membranes were detected using an enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific, Inc.) and visualized using the ChemiDoc system (BioRad Laboratories, Inc.). The intensity of the proteins was measured using ImageJ (edition 1.8.0; National Institutes of Health, Bethesda, MD, US).

Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton-X100 supplemented with 1 mM phenylmethylsulfonyl fluoride before use). Cell lysate in SDS loading buffer (50 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 1% 2-mercaptoethanol, 0.1% bromophenol blue) was boiled and analyzed by SDS-PAGE (Bio-Rad) and transferred to 0.45 μm polyvinylidene fluoride (PVDF) membranes with transfer buffer (25 mM Tris base, 190 mM glycine, 20% methanol, and 80% ddH₂O, pH 8.3). Then the PDVF membranes were blocked with 2% milk (2% bovine serum albumin for phosphorylated antibody) with TBST buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.6, and 0.1% Tween-20) for 1h before incubation with primary antibodies for 2 h at room temperature or 4 °C overnight. After washing with TBST, membranes were incubated with secondary antibody for 1 h and then washed three times with TBST. Cells need to be treated with 50 ng/mL IFN-γ 48 h in advance for phosphorylated protein detection. The antibodies, including anti-Jak2 (Cell Signaling Technology Cat# 3230, RRID: AB_2128522), anti-pJak2 (Cell Signaling Technology Cat# 3771, RRID: AB_330403), anti-STAT3 (Cell Signaling Technology Cat# 12640, RRID: AB_2629499), anti-pSTAT3 (Fluidigm Cat# 3158005, RRID: AB_2661827), anti-TPM4(Abcam Cat# 181085), anti-SOX2 (Abcam Cat# ab92494, RRID: AB_10585428), anti-GAPDH (BioXcell, #BX-008) were used in this study.