Anti areg

SOCS3 expression in Jurkat T cells was detected using Western blotting. Protein extracts were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated with anti-AREG (Santa Cruz Biotechnology, Dallas, TX, USA), anti-TPST-1 (Abcam, Cambridge, UK), and anti-FLNB antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), with anti-GAPDH antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) as a reference. Protein visualization was performed using a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA).

As described previously [20 (link)], cell pellets were lysed in 100 µl RIPA buffer (Roche, Mannheim, Germany) for 15 min at 4 °C. Cell fragments were removed by centrifugation (13,000× g rpm, 10 min, 4 °C), and the supernatants were collected. Then, 40 µg of total RIPA lysates were loaded on polyacrylamide gels. After separation, the gel was blotted onto a PVDF membrane. Each blot was blocked for 1 h with 5% milk powder/TBS-T and incubated overnight at 4 °C with anti-AREG (1:500, Santa Cruz Biotech, Heidelberg, Germany), anti-PML (1:200, Santa Cruz Biotech, Heidelberg, Germany), anti-H3K9 (1:500, Merck KGaA, Darmstadt, Germany), anti-gamma-H2AX (1:1000, Cell Signaling Technology, Frankfurt, Germany), anti-p21 (1:1000, Abcam, Berlin, Germany), or anti-β-actin (1:5000, Sigma-Aldrich) in 3% MP/TBST. After washing three times with TBST, the membrane was incubated with a horseradish peroxidase-coupled secondary antibody (1:2000, anti-rabbit HRP or anti-mouse HRP, Cell Signaling Technology) for 1 h. The immunoreactions were visualized by ECL staining (Bio-Rad, Feldkirchen, Germany).
For competitive Western blot, 400 ng of AREG antibody was pre-incubated with 4 µg of recombinant AREG (Sigma Aldrich, Germany) for 3 h at 4 °C before overnight incubation with the PVDF membrane.

Validation of selected genes (AREG, TPST-1, and FLNB) from RNA sequencing analysis at the protein level was conducted by western blotting. Protein extracts were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated with anti-AREG (Santa Cruz Biotechnology, Dallas, TX, USA), -TPST-1 (Abcam, Cambridge, UK), and -FLNB antibodies (SantaCruz Biotechnology, Dallas, TX, USA) for the target molecule anti-GAPDH antibody (SantaCruz Biotechnology, Dallas, TX, USA) as a reference. Protein visualization was conducted using a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA).