Tbs t

Protein lysates (40 µg per lane) were loaded on a 4–15% SDS-PAGE gel (Bio-Rad), separated in 1× Tris-Glycine Buffer (Bio-Rad), and transferred to PVDF membranes via a wet electro-blotting system (Bio-Rad), all according to the manufacturer’s instructions^{52 (link)}. PVDF membranes were blocked for 1 h in 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T, Bio-Rad), then incubated overnight at 4 °C with antibodies listed in Supplementary Table 2 in 5% bovine serum albumin (BSA) in TBS-T. Blots were then probed with anti-Rabbit IgG conjugated with horseradish peroxidase (1:3000, Cell Signaling Technology) in 5% BSA in TBS-T for 1 h at room temperature. Signal was detected with the Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA), and blot images were collected with a Bio-Rad ChemiDoc imaging system.

Tissue samples (30 μg protein per lane) were subjected to 8% SDS‐PAGE, transferred to nitrocellulose membranes and incubated overnight (4°C) with polyclonal antibody anti‐iNOS1 raised in rabbit, diluted 1:1000 (Merck‐Millipore, Darmstadt, Germany) in PBS‐T (20 mM Tris‐HCl buffer, 150 mM NaCl and 0.05% Tween 20), and with anti‐COX‐2 rabbit polyclonal antibody (Cayman Chemical Co., Ann Arbor, MI, USA) diluted 1:150 in PBS‐T. After several rinses with PBS‐T, membranes were incubated with anti‐rabbit IgG conjugated with horseradish peroxidase, diluted 1:5000 in PBS‐T at RT for 1 hr. The bands were visualized by enhanced chemiluminescence (ECL) and quantified by densitometric analysis. For SMAD3 signalling pathway analysis, 150 μg of total proteins was loaded onto 10% SDS‐PAGE gel, transferred to nitrocellulose membranes, incubated overnight (4°C) with pSMAD3 and SMAD3 [1:1000 in 5% bovine serum albumin (BSA), TBS‐T; Cell Signaling Technology, Danvers, MA, USA] and successively, incubated with anti‐rabbit secondary antibody (1:2000 in 5% BSA in TBS‐T).

Cells were plated onto 12- or 24-well plates, and treated with 5 μg/ml of antibodies for 1 h. After 1 h, 10 μg/ml of zymosan was added. Cells were lysed by adding cold 1 × SDS loading buffer into the wells. Cell lysates were collected, boiled for 5 minutes, and loaded in precast 4–12% gradient gels (Life Technologies). After separation of proteins via electrophoresis, proteins were transferred to PVDF membranes. Blots were blocked with 5% milk in TBST (Cell Signaling) for 1 h. Primary detection antibody was diluted in 5% BSA and TBST for phospho blots and 5% milk and TBST for non-phospho blots. Secondary goat-anti-mouse or anti-rabbit antibodies conjugated to HRP were used for chemiluminescent detection with WesternBright ECL HRP substrate (Advansta). Phospho-ERK antibody (E-4, sc-7383), and ERK 2 antibody (K-23, sc-153) were obtained from Santa Cruz Biotechnology. Phospho-NF-κB p65 (Ser536) (93H1, 3033S) was obtained from Cell Signaling Technology.

The plates were thawed at room temperature, the blocking buffer was discarded, and was treated with serum diluted by 3:1000 in the reaction buffer containing 1x Synthetic block (Invitrogen), phosphate-buffered saline, and 0.1% Tween 20. After the reaction for 1 hour, the plates were washed twice with TBST (#9997; Cell Signaling Technology). Next, the plates were treated with goat anti-human IgG (H+L) Alexa Flour^® 647 conjugate (A21445; Thermo Fisher Scientific) diluted in the reaction buffer for 1 hour, washed twice with TBST, washed again under running reverse osmosis water, and air-dried. Finally, the plates were scanned at a 20-µm resolution using a Typhoon FRA 9500 (GE Healthcare) fluorescent imager. The scanned images were saved as 16-bit tiff files. Array-Pro Analyzer ver. 6.3.1 (Media Cybernetics) was used to record the median by drawing an equal-sized circle around the spots. The negative controls were prepared using distilled water instead of mRNA during protein preparation. The positive controls were prepared using mRNA coding IgG for protein synthesis.

Immunoblot was performed as described previously [24 (link)]. Cells were grown in 150 mm dishes and cultured to 70–80% confluence. Cells were rinsed with PBS, scraped, stored overnight at -80°C, then lysed. Lysates were separated on a 4–15% SDS Page gel (Biorad), and transferred to a nitrocellulose membrane. The membrane was then probed with antibodies diluted in 5% non-fat dry milk in Tris buffered saline (VWR #J640-4L) and 0.1% Tween20 (Fisher Scientific #BP337, TBS-T) and raised against PARP1 (Cell Signaling #9532) that was diluted 1:1000, XRCC1 (Fisher Scientific #MS434P1) that was diluted 1:500, p53 (Santa Cruz Biotechnology #sc-6243) that was diluted 1:1000, BRCA1 (Novus Biologicals #NBP1-45410) that was diluted 1:500, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology #sc-365062) that was diluted 1:1000. The blots were incubated with either horseradish peroxidase (HRP)-labeled secondary antibodies: goat anti-rabbit-HRP or goat anti-mouse-HRP (Cell Signaling Technology). HRP antibody target proteins were detected by incubating with WesternBright Sirius (Advansta).
All immunoblotting was conducted with three biological replicates. Where indicated, protein quantification was conducted with Image Lab Software (BioRad). Band intensity was normalized to loading controls and averaged over the three biological replicates.