P nf κb p65 ser536

Cells were washed twice with ice-cold PBS and solubilized in RIPA buffer (Sigma-Aldrich, Saint Louis, MO) on ice and then was quantified using QuantiPro bicinchoninic acid assay kit (Sigma-Aldrich). Proteins were denatured at 100°C with sample buffer for 5 min. Equal amounts of protein (50 μg) separated by electrophoresis in 10% to 12% SDS-PAGE gels according to their molecular weight. Proteins were transferred onto PVDF membranes (Bio-Rad) and blocked for 2 h in blocking solution (5% nonfat dry milk in TBS containing 0.1% Tween 20). The membrane was then exposed to the primary antibody overnight at 4°C. Snail, slug, NF-κB, p-NFκB P65 (Ser536), STAT3, p-STAT3, AKT, p-AKT were purchased from Cell Signaling (Beverly, MA); Twist1, Zeb1, Zeb2, vimentin, E-cadherin, N-cadherin, β-action were purchased from Santa-Cruz Biotechnology, all primary antibodies dilution is 1:1000. After washing, the membranes were incubated for 1.5 h at room temperature with peroxidase-linked secondary antibody (Santa-Cruz). Signals were revealed with an electrochemoluminescence (ECL) Western Blotting Analysis System (Pierce) using the FluorChem®FC2 (Alpha Innotech, CA), and then quantified using ImageJ® software.

The p-FAK Tyr925 (cat #3284), FAK (cat #3285), p-src Tyr416 (cat #59548), src (cat #2109), p-cofilin Ser 3 (cat #3311), cofilin (cat #5175), NF-κB1 p105/p50 (cat #3035), NF-κB p65 (cat #8242), SNAIL (cat #3879), cleaved PARP (cat #5625), cleaved Caspase-3 (cat #9664), IκBα (cat #4814), Lamin A/C (cat #2032), p-NF-κB p65-Ser536 (cat #3033), and p-IκBα-Ser32 (cat #2859) antibodies were procured from Cell Signaling Technology and were used at 1:1000 dilution; β-actin (cat #sc-47778) antibody was from Santa Cruz Biotechnology and was used at 1:2000 dilution. E-cadherin (cat #610181) was from BD Biosciences and was used at 1:1000 dilution. MIEN1 antibody (cat #H00084299-M02) was from Abnova and was used at 1:2000 dilution. Human recombinant protein EGF was from Gibco (cat #PHG0311). MIEN1 protein was custom produced by Biomatik. The PCR primers were synthesized by Integrated DNA Technologies. The primer sequences are given in Table S2.

TOV21G cells were transfected with siRNA as described earlier. Three days after removal of transfection reagents, the cells were harvested in urea buffer, and extracts were applied on gels, blotted, and used for western detection as described earlier. Antibodies used for immunostaining were pNF‐κB p65 (Ser536) (Cell Signaling, #3033), phospho‐p38 MAP kinase (Thr180/Tyr182) (Cell Signaling, #9215), and Erk1/2 (Cell Signaling #9107), all diluted 1:1000.

Cell lysates were collected using RIPA buffer (Beyotime, Nantong, China) added with a protease inhibitor, and then the different proteins were detected by a standard WB procedure [30 (link)]. The primary antibody to ECM1 (ab126629) was from Abcam (Cambridge, England, UK). The antibodies to p-IKKα/β (Ser176/180, #2697), p-IκBα (Ser32, #2859), NF-κB p65 (#6956), p-NF-κB p65 (Ser 536, #3033), snail (#3879), FAP (#66562) and α-SMA (#19245) were from Cell Signaling Technology (CST, Danvers, MA, USA). The secondary antibodies against mouse (115-035-003) or rabbit IgG (111-035-003) were purchased from Jackson Immuno Research (West Grove, PA, US). The immunoblots were visualized by a chemoluminescence reagent (Millipore, Burlington, MA, USA) and detected on Fluorchem E from Protein Simple (San Jose, CA, USA).

DDX3 rabbit polyclonal antibody (#11115-I-AP) was purchased from Proteintech (China). DDX3 mouse polyclonal antibody (sc-365768) and phospho-PP2A-C mouse monoclonal antibody (sc-271903) were purchased from Santa Cruz Biotechnology (USA). Antibodies for the proteins IKK-β(#2684), p-IKKα/β(S177/181)(#2697), NF-κB p65 (#8242), p-NF-κB p65 (Ser536)(#3033), IκBα(#4814), p-IκBα(#5209), PP2A-C Subunit (#2038), Histone H3(#4499) were purchased from Cell Signaling Technology(U.S.A). β-actin mouse monoclonal antibody (A1978), anti-Flag M2 mouse monoclonal antibody (F1804) and anti-HA rabbit antibody(SAB1306169) were purchased from Sigma-Aldrich. HRP-conjugated goat anti-rabbit or -mouse secondary antibodies were purchased from Jackson ImmunoResearch.

Microglia cells were donated by the Korea Food Research Institute (Wanju, Korea). Chemical reagents were purchased from Sigma-Aldrich (St Louis, MO, USA). RPMI 1640, fetal bovine serum (FBS) and penicillin-streptomycin were obtained from Thermo Scientific Hyclone (Logan, UT, USA). Lipopolysaccharide (LPS) was acquired from Sigma-Aldrich (St Louis, MO, USA). iNOS, COX-2, p-JNK, JNK, p-p38, p38, p-ERK 1/2, ERK 1/2, p-IKKα/β (Ser176/180), IKKβ, p-IκB-α (Ser32), IκBα, p-NF-κB p65 (Ser536), NF-κB p65, HO-1, Nrf2, α-tubulin and Lamin B were purchased from Cell Signaling Biotechnology (Beverly, MA, USA). The antibodies against ionized calcium-binding adapter molecule 1 (IBA-1) and β-actin were obtained from abcam (Cambridge, UK) and Santa Cruz Biotech (Santa Cruz, CA, USA), respectively.