Rabbit anti phospho iκbα

The In-Cell Western assay is a proven method for the rapid quantification of proteins in cells [28 (link), 29 (link)]. BV-2 microglia were seeded into a 96-well plate at 5 × 10⁴ cells/ml, and cells treated at 70% confluence. At the end of each experiment, cells were fixed with 8% formaldehyde solution (100 µL) for 15 min, followed by washing with PBS. The cells were then incubated with primary antibodies overnight at 4 °C. The following antibodies were used: rabbit anti-iNOS (Abcam), rabbit anti-phospho-p65 (Cell Signalling Technology), rabbit anti-phospho-IκBα (Santa Cruz Biotechnology), rabbit anti-NLRP3 (Abcam) and rabbit anti-phospho-p38 (Cell Signalling Technology) antibodies. Thereafter, cells were washed with PBS and incubated with anti-rabbit HRP secondary antibody for 2 h at room temperature. Then, 100 µl of HRP substrate was added to each well and absorbance measured at 450 nm with a Tecan Infinite M microplate reader. Readings were normalised with Janus Green normalisation stain (Abcam).

The in-cell western (cytoblot) analysis is a proven method for the rapid quantification of proteins in cells [18 (link), 19 (link)]. PBMCs were seeded into a black 96-well plate at 5 × 10⁴ cells/mL. At 70% confluence, cells were stimulated with spike glycoprotein S1 (100 ng/ml) for different periods. At the end of each experiment, cells were fixed with 8% paraformaldehyde solution (100 μL) for 15 min, followed by washing with PBS. The cells were then incubated with primary antibodies overnight at 4 °C. The following antibodies were used: rabbit anti-phospho-p65 (Cell Signalling Technology), rabbit anti-phospho-IκBα (Santa Cruz Biotechnology), rabbit anti-total IκBα (Santa Cruz Biotechnology), rabbit anti-phospho-p38 (Cell Signalling Technology) and rabbit anti-NLRP3 (Abcam) antibodies. 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 each well and absorbance measured at 450 nm with a Tecan Infinite M microplate reader. Readings were normalised with Janus Green normalisation stain (Abcam).

BV-2 cells were treated with skimmianine (10, 20 and 30 μM) for 30 min, followed by stimulation with LPS (100 ng/mL) for different time points. Immunoblotting was carried out on 20–40 μg of cell lysates which were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The membranes were incubated with blocking buffer at room temperature for 1 h and further incubated with primary antibodies overnight at 4 °C. The primary antibodies used were rabbit anti-COX-2 (1:1000; Abcam, Cambridge, UK), rabbit anti-iNOS (1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-IκBα (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-phospho-p65 (1:500; Abcam), and rabbit anti-actin (1:1000; Sigma). Thereafter, membranes were washed in tris-buffered saline + Tween 20 (TBS-T), followed by incubation with Alexa Fluor 680 goat anti-rabbit secondary antibody (1:10,000; Thermo Scientific, Waltham, MA, USA) at room temperature for 1 h. Blots were detected using a Licor Odyssey Imager. All Western blot experiments were carried out at least three times, and blots were quantified using Image J software (Version 9).