Rabbit anti phospho p65

The in-cell western is now an established method for the rapid quantification of proteins in cells (Velagapudi et al. 2019b (link)), because it combines the specificity of western blotting with the quantification capability of ELISA. In these experiments, RAW264.7 macrophages were seeded into a 96-well plate (5 × 10⁴ cells/mL). At 70% confluence, cells were treated with MTC (5–20 μM) for 30 min, followed by stimulation with LPS (1 µg/mL) and IFNγ (10 ng/mL) for different incubation periods. Cells were fixed with 8% paraformaldehyde solution (100 μL) for 15 min. and then washed with PBS. The cells were then incubated with the primary antibodies overnight at 4 °C. The following antibodies were used: rabbit anti-COX-2 (Abcam), rabbit anti-iNOS (Abcam), rabbit anti-phospho-p65 (Cell Signalling technologies), rabbit anti-phospho-IκB (Santa Cruz Biotechnology), rabbit anti-IκB (Santa Cruz Biotechnology), and rabbit anti-phospho-AMPKα (Santa Cruz Biotechnology). Thereafter, cells were washed with PBS and incubated with anti-rabbit HRP secondary antibody for 2 h at room temperature. Then, 100-µL HRP substrate was added to the plate and signal measured at 450 nm with a microplate reader. Readings were normalised with Janus Green stain (Abcam).

For MDA‐MB‐231 cells, the cell seeding and the treatments were carried out as indicated above. Shortly, the cells were transfected with siRNAs (siFn14 or siCtrl, Dharmacon) and treated with DAPT (or DMSO) in 1% FCS media 1 day before the experiment. The treatment with 100 ng/ml human recombinant TWEAK (Biotechne) or 10 ng/ml human recombinant TNF (Biotechne) were carried out in the indicated time points. The cells were lysed with RIPA buffer (150 mM NaCl, 10 mM Tris–HCl pH 8.0, 2 mM EDTA, 1% Triton‐X100, 0.1% sodium deoxycholate, 0.1% SDS) containing protease inhibitor and phosphatase inhibitor PhosStop (Roche). Lysates were loaded on Schägger gels or 12% SDS‐gel and immunoblotted with the following antibodies: rabbit anti‐Fn14 (Cell Signaling, Cat #4403), rabbit anti‐IκBa (Cell Signaling, Cat #9242), mouse anti‐phospho(Ser32/36) IκBa (Cell Signaling, clone 5A5, Cat #9246), rabbit anti‐P65 (Cell signaling, clone D14E12, Cat #8242), rabbit anti‐phospho P65 (Cell Signaling, clone 93H1, Cat # 3033), mouse anti‐β‐actin (Sigma, Cat A5316), and rabbit anti‐calnexin (Enzo, Cat ADI‐SPA‐860).