Anti ets1

ChIP-PCR assays were performed by Simple ChIP plus Enzymatic Chromatin IP Kit (Cell Signaling: #9005) according to the manufacturer’s protocol. Briefly, cells were cross-linked with 1% of formaldehyde and lysed for nuclei preparation. Nucleus pellet was treated with Micrococcal nuclease and sonicated for chromatin fragmentation. Protein–chromatin complex was incubated with antibodies targeting anti-NFATc2 (ab2722), anti-NFKB1 (ab7971), anti-RELA (ab7970), anti-DNMT3A (ab13888), anti-DNMT3B (ab13604), anti-H3K4me1 (ab8895), anti-H3K4me3 (ab8580), anti-H3K9me3 (ab8898), anti-Ets1 (Cell Signaling: #14069), anti-H3Ac (Merck: 06–599), or anti-H3K27me3 (Merck: 07–449) at 4 °C for overnight. Rabbit or mouse IgG (Vector Laboratories) was used as negative control. After immunoprecipitation, 50 μl of Dynabeads protein G or A (Life technologies, Oslo) were added and rotated further for 6 h at 4 °C. Ab/protein/chromatin complexes were reverse-cross-linked at 65 °C overnight and DNA was purified by DNA purification columns (Cell Signaling: #10010). The relative enrichment of specific regions of precipitated DNA were measured by real-time PCR (qPCR). To quantify protein binding in specific genomic locus, purified DNA was used for real-time PCR. Primer sequences are listed in Supplementary Table 2. H3K27Ac, and H3K27me3 ChIP-seq data set (GSE38548) were re-analyzed^{35 (link)}.

Whole cell lysates were prepared by extracting proteins using RIPA buffer containing 50 mM Tris (pH 7.4), 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na₃VO₄, 1 mM NaF, 1 g/mL pepstatin, and 1 g/mL aprotinin. Western blot analysis was performed as previously described [13 (link)]. The protein concentration was measured with a Bradford assay (Bio-Rad, Hercules, USA), and an equal amount of each lysate was separated on a 10% SDS-polyacrylamide gel. After the proteins were transferred to polyvinylidene fluoride membranes (Millipore, MA, USA), the membranes were blocked for 1 h in TBST containing 5% skimmed milk. Blocked membranes were then incubated with primary antibodies (goat anti-type I collagen (#1310); Southern Biotechnology, Birmingham, AL, anti-phospho-ERK (#4377), anti-ERK (#9102) and anti-Ets-1 (#14069), anti-phospho-Smad2 (#3108), anti-Smad2 (#8685); Cell Signaling Technology, MA, USA,) diluted 1:1000 overnight at 4°C, washed three times with TBST for 15 min, and incubated with horseradish peroxidase-conjugated anti-goat or anti-rabbit secondary antibody (Sigma Aldrich, St. Louis, USA) diluted 1:5000 for 1 h at room temperature. The membranes were visualized using an ECL detection kit (Thermo Fisher Scientific).

Western blotting was performed using standard methods, and the following antibodies were used for analysis: anti-NFkB2 (Cell Signalling; 3017), anti-ETS1 (Cell Signalling; 14069), anti-IRE1α (Santa Cruz; 390960) and anti-GAPDH (Santa Cruz; 32233). Antibody dilutions used were 1:10000 for anti-GAPDH and 1:1000 for the other antibodies.

Cell pellets were lysed with RIPA buffer supplemented with proteinase inhibitors and phosphatase inhibitors on ice for 30 min and centrifugated at 12,000 rpm at 4 °C for 10 min. Total protein concentration was measured by Bradford assay, and equal amounts of protein were resolved by SDS-PAGE and transferred onto the PVDF membrane. The membrane was blocked with 5% BSA in TBST and then incubated with primary antibodies at 4 °C overnight. An appropriate secondary antibody was incubated at room temperature for 2 h. The immunoreactivities were detected by the Amersham ECL Prime Western Blotting Detection Reagent (Cytiva) and captured by a chemiluminescence imaging system (Bio-Rad). The following primary antibodies were commercially obtained: anti-PARP (#9532), anti-caspase-3 (#9662), anti-cleaved-caspase-3 (#9661), anti-ETS-1 (#14069), anti-ACTIN (#4970), anti-GPX4 (#52455), anti-p-AKT (#4060), anti-AKT (#4691), anti-ERK (#4695), and anti-p-ERK (#8544) from Cell Signaling Technology; anti-SLC7A11 (A2413) and anti-ACSL4 (A16848) from Abclonal.