Nitrocellulose blotting membrane

Cells cultured in 3D and 2D were harvested and lysed in ice-cold RIPA (radio immunoprecipitation assay) buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors (Sigma). Lysates were purified by centrifugation at 12,000 rpm for 10 minutes at 4°C and supernatant was collected. Purified lysates were boiled in 1x Laemmli sample buffer (0.04 M Tris-HCl pH 6.8, 0.2% SDS, 0.01% bromophenol blue, 10% β-mercaptoethanol and 10% glycerol) for 5 minutes at 95°C. Aliquots of cell lysates containing equal protein mass were resolved by 10% SDS-PAGE gels, transferred to nitrocellulose blotting membranes (GE Healthcare Life Sciences), blocked with 5% skim milk (w/v) in TBS (20 mM Tris, 150 mM NaCl, pH 7.5), 0.1% Tween 20, for 1 hr at room temperature and probed with primary antibodies at the recommended dilutions for overnight incubation at 4°C. Subsequently, membranes were probed with relevant secondary antibodies conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories) for 1 hr at room temperature. After washing, western blot membranes were developed using chemiluminescent substrate for detection of HRP (Millipore) and proteins were detected by chemiluminescence (Fujifilm LAS-4000). Quantification of the mean pixel density of the protein bands was determined using NIH ImageJ plugin.

RAW264.7 cells were treated with dead or live E. tarda as above for 0 h, 1 h, 2 h, and 4 h. The cells were lysed on ice for 30 min with RIPA lysis buffer (Beyotime, Beijing, China) containing phosphatase inhibitor cocktail (Beyotime, Beijing, China). The cell lysates were mixed with SDS-PAGE loading buffer and boiled at 100 °C for 10 min. The samples were then subjected to SDS-PAGE, and the separated proteins were electro-transferred from gels to nitrocellulose blotting membranes (GE healthcare, Germany). The membranes were soaked in TBST (20 mM Tris, pH 7.5; 500 mM NaCl; 0.1% Tween 20) containing 5% bovine serum albumin (Solarbio, Beijing, China) for 2 h. The membranes were incubated with rabbit anti-phospho-NF-κB p65 (Ser536) monoclonal antibody (Cell Signaling Technology, Beverly, MA, USA), anti-NF-κB p65 monoclonal antibody (ABclonal, Wuhan, China), or anti-β-actin monoclonal antibody (ABclonal, Wuhan, China) at 4 °C for overnight. After extensive washing with TBST, the membranes were incubated with HRP-conjugated anti-rabbit antibody (Abcam, Cambridge, UK) for 1h at room temperature. The membranes were washed with TBST for three times and incubated with enhanced chemiluminescence (ECL) solution (Beyotime, Beijing, China). The membranes were visualized using a GelDoc XR System (Bio-Rad, Hercules, CA, USA).