Rabbit anti iκb

After various treatments, the cells were washed with PBS and lysed with RIPA lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02 % sodium azide, 0.1 % sodium dodecyl sulfate (SDS), 1 % Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF)). Equal amounts of protein from the different treatment groups were separated by SDS-polyacrylamide gel electrophoresis (10 % gel) and transferred to nitrocellulose membranes (Amersham Biosciences). The membranes were blocked with 4 % skim milk in Tris-buffered saline Tween-20 (TBST) and then incubated with primary antibodies (goat anti-PTP1B (1:500 dilution, Santa Cruz Biotechnology), polyclonal rabbit anti-phospho- or total forms of Src (1:1000 dilution, Cell signaling), rabbit anti-IκB (1:1000 dilution, Santa Cruz), mouse anti-β-actin (1:2000 dilution, Sigma-Aldrich), mouse anti-α-tubulin (1:5000 dilution, Sigma-Aldrich), and goat anti-LCN2 (1:500 dilution, R&D systems)). After thorough washing with TBST, horseradish peroxidase-conjugated secondary antibodies were applied. The blots were developed using an enhanced chemiluminescence detection kit (SuperSignal™ West Femto, ThermoFisher).

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).