Anti phosphorylated jak2

A549 and H520 cells were harvested and lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology). The BCA assay kit (Bio-Rad Laboratories, Inc.) was used to determine protein concentration. The protein samples (40 μg per lane) were loaded and electrophoresed via SDS-PAGE on a 10% gel, and then transferred to PVDF membranes (EMD Millipore). Membranes were blocked with 5% skimmed milk at room temperature for 1 h, and subsequently co-incubated with the primary antibodies at 4˚C overnight. Subsequently, the bound primary antibodies were incubated with the corresponding secondary antibodies (1:5000, cat. no. ab6721, Abcam) overnight at 4˚C, and then detected by enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.). The primary antibodies used were purchased from Abcam and were as follows: Anti-KLF4 (1:1000; cat. no. ab215036; Abcam), anti-MSI2 (1:2000; cat. no. ab76148; Abcam), anti-JAK2 (1:1000; cat. no. ab108596; Abcam), anti-phosphorylated (p)-JAK2 (1:1000; cat. no. ab32101; Abcam), anti-STAT3 (1:2000; cat. no. ab68153; Abcam), anti-p-STAT3 (1:1000; cat. no. ab76315; Abcam), anti-Wiskott-Aldrich syndrome protein family member 3 (WASF3; 1:2000; cat. no. ab68031; Abcam) and anti-GAPDH (1:1,000; cat. no. ab9484; Abcam). ImageJ software (version 2.0; National Institutes of Health) was used for semi-quantification of the band densities.

Total protein from HTC116 cell lines was extracted using a mammalian protein extraction kit (cat. no. CW0891M; CoWin Biosciences) and used according to the manufacturer's instructions. Total protein was quantified using a bicinchoninic acid assay kit (cat. no. CW0014S; CoWin Biosciences). The mass of protein loaded per lane were 10–40 µg on a 12.5% SDS-PAGE gel. The proteins were transferred to PVDF membranes. The membranes were blocked with 5% no-fat milk at room temperature for 1 h. The membranes were incubated with the following primary antibodies overnight at 4°C: Anti-PD-L1 (1:2,000; cat. no. 13684; Cell Signaling Technology, Inc.), anti-JAK2 (1:2,000; cat. no. ab39636; Abcam), anti-phosphorylated (p)-JAK2 (1:2,000; cat. no. ab32101; Abcam), anti-STAT1 (1:2,000; cat. no. ab31369; Abcam), anti-p-STAT1 (1:1,000; cat. no. ab109461; Abcam) and anti-GAPDH (1:1,000; cat. no. ab181602; Abcam). The membranes were incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody at room temperature for 25 min (1:10,000; cat. no. ab6721; Abcam). Protein bands were visualized using ECL reagents (iBright cat. no. CL750; Thermo Fisher Scientific, Inc.) and a ChemiDoc XRS + imaging system (Bio-Rad Laboratories, Inc.).