Anti stat1 antibody

In brief, 1 × 10⁷ A549 cells were treated with 20 ng/mL of IFNγ for 1.5 h or sequentially treated with 20 ng/mL of IFNγ for 2 h with 20 ng/mL of EGF for 1.5 h. After treatment, A549 cells were fixed by 1% formaldehyde and fragmentized by sonication. A549 cells were resuspended and incubated with control IgG, anti-STAT3, and anti-STAT1 antibodies (Cell Signaling, Danvers, MA, USA) at 4 °C overnight for immunoprecipitation. After incubation, the antibodies were captured by Dynabead-Protein A (Life Technologies, Waltham, MA, USA). The Dynabeads were washed and consequently pulled down by a Sample Magnetic Rack. DNA fragments were eluted by boiling the Dynabeads and concentrated by a FavorPrep GEL/PCR Purification Mini Kit (Favorgen Biotech Corp., Wembley, WA, Australia). The DNA preparation was analyzed by real-time PCR using Fast SYBR Green Master Mix (Applied Biosystem, CA, USA) with primer pairs shown in Table 1. The primer sequences amplify the PDL1 and MCL1 promoter predicted from PROMO (http://alggen.lsi.upc.es/, accessed on 4 May 2021).

HCT116 cells were treated with 5 μM Δ⁹-THC or 5 μM Δ⁹-THC plus 50 μM BODIPY-THIF for 6 h, cross-linked with 2% formaldehyde for 15 min, collected by scraping in PBS and then centrifuged and lysed in 1 mL of IP buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.5, 5 mM EDTA, 0.5% Nonidet P-40 and 1% Triton X-100) containing protease inhibitors. The obtained nuclear pellet was resuspended in IP buffer and sonicated. ChIP assays using control rabbit IgG (Santa Cruz) or anti-STAT1 antibodies (Cell Signaling Technology, Danvers, MA, USA) were performed. The immunoprecipitated DNA and input DNA were extracted by incubating the samples with 100 μL of 10% Chelex (Bio-Rad Laboratories, Hercules, CA, USA), then boiling them to reverse the cross-linking and then centrifuging them to remove the Chelex slurry. PCR was performed using two oligonucleotide primers as follows: GDNF (forward primer, 5’-CAGCATGGAAATGAAGCCTA-3’; reverse primer, 5’- TAGTTTAGTCCCCAGGCTAG-3’); IGFBP6 (forward primer, 5’- TGCTGACAATGAGGTTCGTAT-3’; reverse primer, 5’- GTTATGCAACAGGGACCATC-3’); IGF2 (forward primer, 5’- CTGAATTCTCTAGAACGGGCATTCAGCA-3’; reverse primer, 5’- GGGGGCAGGGAGCCGCAGAG-3’); SCF (forward primer, 5’-ATAGGCTAGCAGCACAGACTTCCCTCCACAAAGT-3’; reverse primer, 5’-CATGGAAGCTTTGTGGCGACTCCGTTTAGCT-3’); and VEGFA (forward primer, 5’-GCGTGTCTCTGGACAGAGTTT-3’; reverse primer, 5’- AGCCTCAGCCCTTCCACA-3’).

Tissue and cell extracts were analyzed with the following primary antibodies: anti-collagen I, anti-α-SMA, anti-MyD88 (Abcam, Cambridge, Cambs, UK), anti-p-JAK1/2, anti-JAK1/2, anti-p-STAT1, anti-STAT1 antibodies (Cell Signaling, Danvers, MA, USA) and β-actin (Santa Cruz Biotechnology, CA, USA). HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were used as secondary antibodies. Blots were scanned using a Clinx Science Instrument.