Anti actin

Cells were trypsinized, pelleted and washed twice with cold phosphate-buffered saline (PBS). Cell pellets were then lysed in cold low-ionic-strength buffer (50 mM NaCl, 1% IGEPAL, 10% glycerol, 10 mM HEPES, pH 7.4) with fresh added proteinase inhibitor cocktail, DTT and PMSF (ThermoFisher Scientific, San Jose, CA, USA). After the determination of protein concentration, samples were denatured in SDS sample buffer, sonicated three times for 15 s each time (Branson150) and centrifuged at 10,000 rpm for 20 min. Supernatants were heated at 95 °C for 5 min. Proteins were resolved using Criterion TGX 4–15% precast PAGE gels and transferred to nitrocellulose membranes with a Bio-Rad Trans-Blot Turbo transfer system. Primary antibodies used were anti-RelA (ab194726, Abcam, Waltham, MA, USA; MAB5078, R&D, Minneapolis, MN, USA); anti-actin (937215, R&D) was used as a loading control. Blots were imaged and quantified.

Cell lysates were collected using sample buffer solution with reducing reagent (6×) for SDS/PAGE (Cat. No. 09499‐14; Nacalai Tesque, Kyoto, Japan), then electrophoresed onto a 10% SDS/PAGE, and blotted onto a poly(vinylidene difluoride) membrane. The membranes were incubated with anti‐nephronectin (Cat. No. AF4298; R&D Systems, Minneapolis, MN, USA) and anti‐actin (Cat. No. A5060; MERCK, Darmstadt, Deutchland) as the first antibodies and then further probed with anti‐mouse IgG horseradish peroxidase‐linked (Cat. No. NA931V; GE Healthcare, Little Chalfont, UK) and anti‐goat IgG horseradish peroxidase‐linked (Cat. No. NB7352; NOVUS, Littleton, CO, USA) secondary antibodies. Proteins were visualized using ECL Prime Western Blotting Detection reagent (Cat. No. #RPN2232; GE Healthcare).

Cells were trypsinized, pelleted and washed twice with cold phosphate-buffered saline (PBS). Cell pellets were then lysed in cold low ionic strength buffer (50 mM NaCl, 1% IGEPAL, 10% Glycerol, 10 mM HEPES, pH7.4) with fresh added proteinase inhibitor cocktail (1:100 vol/vol dilution, Sigma P8340), 1 mM DTT and 1 mM PMSF (Sigma P7626). After determination of protein concentration, samples were denatured in SDS sample buffer, heated at 95°C for 5 min, and fractionated using Criterion TGX 4-15% PAGE gels. Proteins were transferred to PVDF membranes and probed with anti-IRF1 (Proteintech 11335-1-AP, 1:500), anti-FLAG M2 (Sigma F1804, 1:1000) or anti-actin (937215, R&D, 1:5000) antibodies (Ab). Anti-TBP Ab (Biolegend 668306, 1:1000) was used as a loading control. Blots were imaged and bands quantified.