Horseradish peroxidase labeled secondary antibody

Cells were washed in ice-cold PBS and lysed in RIPA buffer (10 mM Tris-HCl (pH 7.2), 160 mM NaCl, 1% Triton X-100 (Sigma-Aldrich), 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM EDTA and 1 mM EGTA) containing 40 μl/ml complete protease inhibitors (Roche Applied Science). Lysates were cleared by centrifugation at 14 000×g for 10 min. Equal amounts of proteins were electrophoretically separated on 10% NuPAGE Novex Bis-Tris gels (Life Technologies—Invitrogen) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk in PBS, and probed with antibodies toward actin (1 : 2000, MP Biomedicals 691001, Santa Ana, CA, USA and 1 : 1000 sc-81178), caspase-3 (1 : 500, Pharmingen 67341A), caspase-8 (1 : 500, Cell Signaling 97465), cIAP1 (1 : 200, R&D AF8181, Minneapolis, MN, USA), cIAP2 (1 : 300 NB110-57030), p52/p100 (1 : 300, Cell Signaling #4882), Rel B (1 : 300 sc-48366), p65 (1 : 400 sc-372), α-tubulin (1 : 400 sc-5286), lamin B (1 : 400 sc-6216) and RIP1 (1 : 300 BD Trans Lab 610458, Franklin Lakes, NJ, USA), followed by incubation with a horseradish peroxidase-labeled secondary antibody (1 : 5000 GE Healthcare, Little Chalfont, UK). The chemiluminescence was detected with a charge-coupled device camera (Fujifilm, Minato, Japan).

Cells were lysed in 1% NP-40, 137 mM NaCl, 2 mM EDTA, and 20 mM tris-HCl (pH 8) with protease inhibitor cocktail (Roche, 04693116001) and assayed for protein concentration by the BCA assay (Pierce, 23225). Equal protein amounts were added to Lithium dodecyl sulfate (LDS) loading buffer (Bio Rad 161-0747) containing 10% β-mercaptoethanol and run on 4 to 12% gradient SDS–polyacrylamide gel electrophoresis gels. Proteins were transferred onto 0.2-μm-pore polyvinylidene difluoride membranes (Merck, IPVH00010). Nonspecific background was blocked in 5% (w/v) dry skimmed milk (Sigma-Aldrich) in PBS containing 0.1% Tween 20, and membranes were incubated with the primary antibody (Abcam, ab53015) overnight at 4°C, followed by 1 hour at room temperature in the appropriate horseradish peroxidase–labeled secondary antibody (GE Healthcare). Blots were developed with enhanced chemiluminesence Clarity (Bio-Rad, 1705061). Intensity was quantified using ImageJ. In mouse samples, the ratio of pro to active MMP3 was used to express the percentage of active MMP3, but the ratio of active MMP3 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for human samples because no pro-MMP3 was detected in some samples.

RGM-1 cells and rat gastric mucosal tissues were homogenized with RIPA lysis buffer containing protease inhibitors to prepare the protein solutions, and Western blot was performed to analyze the protein expression as previously described [31 (link)]. Briefly, proteins were transferred from the gels onto polyvinylidene difluoride (PVDF) membranes after separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The PVDF membranes were then blocked with PBS containing 5% non-fat milk and 0.1% tween 20, followed by incubations with a series of primary anti-bodies against COX-1, COX-2, iNOS, NF-κB and TNF-α (1:1000) at 4 °C overnight. After washing with PBST (PBS with 0.1% tween 20), the membranes were incubated with horseradish peroxidase-labeled secondary antibody (1:5000 dilution, General Electric, Boston, MA, USA) at room temperature for 1 h. Next, the signals of the bands were developed using Amersham^TM ECL Select^TM Western Blotting Detection Reagent. The fluorescent bands were then detected by Luminescence Image system (M2-8068, Hansor, Taichung, Taiwan), and their densitometry intensities were quantified using ImageJ software.