Anti mouse antibody

Choline chloride (Salus Nutra Inc., Xian, China), DL-malic acid (Salus Nutra Inc., Xian, China), commercial chitin (Carbosynth, China), sucralfate (Combiphar, Bandung, Indonesia), ethanol, pure 99.5% (Merck Millipore-Sigma Aldrich, USA), ethyl ether (Merck Millipore-Sigma Aldrich, St. Louis, MI, USA), carboxymethyl cellulose (CMC) (BrataChem, Bandung, Indonesia), Bouin solution, distilled water (BrataChem, Bandung, Indonesia), mouse monoclonal IgG1 NF-kappaB p65 antibody (Santacruz Biotechnology Inc., K2320, Shanghai, China), β-actin antibody (Thermo Scientific MA5-15739, Singapore), anti-mouse antibody (Li-Cor C90408-07, PT. Elo Karsa Utama, Jakarta, Indonesia), bovine serum albumin (BSA Protein, Thermo Scientific, Singapore), sodium dodecyl sulfate (SDS) gel 15% (Merck Millipore-Sigma Aldrich, St. Louis, MI, USA), polyvinylidene difluoride (PVDF) membrane (Merck Millipore-Sigma Aldrich, St. Louis, MI, USA), phosphate buffer saline-tween 20 (PBST) 0.1%, protein ladder (Thermo Scientific, Singapore), running buffer, transfer buffer, lysis buffer.

RPPA analysis was performed using a procedure previously optimized and validated [61 (link)]. Briefly, cell lysates obtained from 800,000 cells (ø 10 cm Petri dishes) were solubilized in 4×SDS loading buffer (Sigma-Aldrich, Milano, Italy) and heated for 5 minutes at 95°C. Samples were spotted in duplicates onto nitrocellulose-coated glass slides (Grace Bio-labs, Bend, OR, USA) using a microarraying robot (MicroGrid 610, Digilab, Marlborough, MA, USA). The printed slides were blocked and incubated overnight at 4°C with shaking with the specific primary antibodies (Supplementary Table 3) (Cell Signalling Technology, Danvers, MA, USA). β-actin was included as a house-keeping protein to control protein loading. After incubation with infrared secondary antibodies (800 CW LI-COR anti-rabbit antibody and 700 CW LI-COR anti-mouse antibody), the slides were scanned with a Licor Odyssey scanner (LI-COR, Biosciences, Lincoln, NE, USA) at 21 μm resolution at 700 and 800 nm. The fluorescent data were processed with GenePix Pro-6 Microarray Image Analysis software (Molecular Services Inc., Sunnyvale, CA, USA). Protein signals were determined with background subtraction and normalization to the internal housekeeping targets using an RPP analyzer.

Lysates were solubilised in 4 × SDS loading buffer and heated for 5 min at 95 °C. RPPA assays of the components of the NF-κB, PI3K–Akt, MAPK, JAK–STAT–c-Sr, and inflammasome (NALP1) signalling pathways were performed as previously described [10 (link)].
Briefly, samples were spotted in duplicates onto a nitrocellulose-coated glass slide (Grace Bio-labs, Bend, OR, USA) using a microarray robot (MicroGrid 610, Digilab, Marlborough, MA, USA). The printed slides were blocked and incubated with specific primary antibodies listed in Table 2 (Cell Signalling) overnight at 4 °C, with shaking. Β actin was included as a housekeeping protein, to control protein loading. After incubation with infrared secondary antibodies (800 CW LI-COR anti-rabbit antibody and 700 CW LI-COR anti-mouse antibody), the slides were scanned with a Licor Odyssey scanner (LI-COR, Biosciences) at 21 um resolution at 700 nm and 800 nm. The fluorescent data were processed with GenePix Pro-6 Microarray Image Analysis software (Molecular Services Inc., San Diego, CA, USA). Protein signals were determined with background subtraction and normalisation to the internal housekeeping targets using the RPP analyser [44 (link)].