P stat3 antibody

After IR, the RIPA lysis buffer was used to lyse the cells. After the protein concentration was confirmed by the BCA protein detection kit (Beyotime, Shanghai, China), the proteins of the same concentration were separated using 10% SDS-PAGE and then transferred to PVDF membrane (Millipore, USA). Following the blocking step using 5% milk, antibodies were added to the membranes overnight at 4°C, including IL-32 antibody from Proteintech, USA, and STAT3 antibody, p-STAT3 antibody, Bax antibody, and GAPDH antibody from Cell Signaling Technology, USA. Subsequently, the membranes were incubated with the secondary antibodies (Cell Signaling Technology, USA) at room temperature for 2 hours. Immunoblotting protein was detected by enhanced chemiluminescence.

Western blot experiments were performed as previously reported (Xue et al., 2017 (link); Qin et al., 2018 (link)). In brief, 3 × 10⁵ cells were cultured in 6-cm dishes treated with specific concentrations of SDL-1 (0, 10, 20, and 40 μM) for 24 h. And then all cells were lysed in RIPA lysis buffer (Absin Bioscience Inc., Shanghai, China) containing protease inhibitor mixture (PMSF). The cell lysates were quantified by the BCA Protein Assay Kit (Absin, Shanghai, China). Equal amounts of protein were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, United States). Block the membranes in 5% skim milk at room temperature and incubated with primary antibody overnight at 4 °C followed by incubation with appropriate secondary antibody at room temperature. Antibodies used are as follows: STAT3 antibody (Cell Signaling Technology, #12640S), p-STAT3 antibody (Cell Signaling Technology, #49081S), and GADPH antibody (Proteintech, #60004-1-Ig). The protein bands were detected by ECL Chemiluminescent Substrate Reagent Kit (Biosharp, Hefei, China) and scanned using the ImageQuant 800 (Amersham, United Kingdom).

To analyze the pSTAT3 binding ability, we added 5 μg of pSTAT3 antibody (Cell Signaling Inc., Danvers, MA) to perform chromatin immunoprecipitation (ChIP) (Merck). Two microliters of ChIP samples were used for qPCR analysis, as described previously,³⁷ using gene‐specific primers (Table 1). For the ChIP data, we adjusted the signals to IgG control. To analyze the ChIP‐qPCR, the Ct minus the Ct from input control to obtain the ΔCT, and then the ΔCT minus the ΔCt from negative control IgG to obtain the ΔΔCT. The fold change was converted from ΔΔCT by 1.94ˆ(ΔΔCt).

Liver tissues extracts were prepared from liver cancer tissue specimens and the cell protein from HUVEC treated with the RIPA buffer. Then, an equal amount of protein (50 μg) was loaded onto 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed dried milk buffered for 2 hours at 25°C and incubated with primary antibodies against STAT3 rabbit mAb (dilution, 1 : 1,000; cat. no. 9959; Cell Signaling Technology, Inc., Danvers, MA, USA), p-STAT3 antibody (dilution, 1 : 1,000; cat. no. 9914; Cell Signaling Technology, Inc.), and PCNA antibody (dilution, 1 : 1,000; cat. no. 2586; Cell Signaling Technology, Inc.), overnight at 4°C. Next, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody for 2 hours at 25°C. The membranes were washed four times with tris-buffered saline with Tween-20 and detected by a fluorescence visible imaging system BIO-RAD imaging system (BIO-RAD, Hercules, CA, USA). The intensity of each band was evaluated by the densitometry test using Image Pro Plus 4.5 software (Media Cybernetics, Inc., Bethesda, MD, USA). The quantification was normalized to the comparable value of GAPDH expression.

ChIP assay was performed with the EZ-Magna ChIP A/G (17–10,086, Millipore) and a p-STAT3 antibody (5 μg per reaction; 9131, Cell Signaling Technology, Boston, MA, USA) in accordance with the manufacturer’s instruction. ChIP-derived DNA was quantified using qRT-PCR to detect enrichment of chromatin. The sequences of the primers were as follows: for the − 668 site of HEGBC promoter, 5’-CACACTGGATTTGTTTCTG-3' (forward) and 5′-GGGTGGTTGGGTTTTTTTT-3' (reverse); for the − 930 site of HEGBC promoter, 5’-CTGCCAACCTGGAAGAAA-3' (forward) and 5’-TTAGGGATTAGGAACCCC-3' (reverse); for the − 1211 site of HEGBC promoter, 5’-ATGTAGTATCATGAGCCTGGG-3′ (forward) and 5’-GCAAAGTTATGGAAGCCGTG-3′ (reverse); for the − 1556 site of HEGBC promoter, 5’-GCAAAGAGAGGCAGGAGT-3′ (forward) and 5’-TGCTGGGTAAATGAGGACA-3′ (reverse); for the distal non-binding site (negative control, NC) of HEGBC promoter, 5’-GTTGTCTCATTGTGTCCC-3′ (forward) and 5’-TGTGTGTTTTTCCCTCTTG-3′ (reverse).