Anti ikkα

We purchased pyridostigmine bromide (PB), permethrin (Per), Neomycin trisulfate hydrate, Enrofloxacin, and Ribavirin from Sigma-Aldrich (St. Louis, MO, USA). Anti-claudin-2, anti-MyD88, anti-MCP-1, and anti-β-actin primary antibodies were purchased from Abcam (Cambridge, MA, USA). anti-β-tubulin, anti-TLR3, anti-TLR7, anti-IKKα, anti-p65, and anti-IL6 primary antibodies were purchased from Santacruz Biotechnology (Dallas, TX, USA) while anti-TRAF6 was purchased from Abclonal Technology (Woburn, MA, USA). Species-specific biotinylated conjugated secondary antibodies and Streptavidin-HRP (Vectastain Elite ABC kit) were purchased from Vector Laboratories (Burlingame, CA, USA). Fluorescence-conjugated (Alexa Fluor) secondary antibodies and ProLong Diamond antifade mounting media with DAPI were purchased from Thermofisher Scientific (Grand Island, NY, USA). All other chemicals used in this study were purchased from Sigma unless otherwise specified. Animal tissues were paraffin-embedded and sectioned into slides by AML laboratories (Baltimore, MD, USA).

Total protein from OSCC tissue samples and cells was extracted using RIPA lysis buffer containing 1% protease inhibitor (Sigma). 50ug of protein was loaded on a 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and separated by electrophoresis. Then, the protein was transferred to polyvinylidene fluoride membranes (Merck Millipore). After blocking non‐specific binding sites with 5% non‐fat milk, the membranes were incubated with the following primary antibodies: anti‐IKKα (sc‐7606, 1:500; Santa Cruz Biotechnology), anti‐Naa10p (sc‐3739201:1000; Santa Cruz), anti–E‐cadherin (9782, 1:1000; Cell Signaling Technology, CST), anti‐Snail (9782, 1:1000; CST), anti‐Slug (9782, 1:1000; CST), anti–Claudin‐1 (9782, 1:1000; CST), anti‐Smad2 (5339, 1:1000; CST), anti–p‐Smad2 (3108, 1:1000; CST), anti‐Smad3 (9523, 1:1000; CST), anti–p‐Smad3 (9520, 1:1000; CST), anti–β‐Actin (TA09, 1:1000; ZSGB‐BIO, Beijing, China) overnight at 4℃. Next, the membranes were washed with TBST and incubated with peroxidase‐conjugated IgG antibody at room temperature for 2 hours, and the immune complex was visualized by using enhanced chemiluminescence (34094; Pierce).

Dorsolateral prostate tissue from treated and control groups were subjected to preparation of total tissue lysate and isolation of cytosolic and nuclear fractions as described previously [38 (link)]. For Western blotting, 25 μg of protein was resolved over 4–20% Tris-glycine polyacrylamide gel and then transferred onto the nitrocellulose membrane. The blots were blocked using 5% non-fat dry milk and probed using appropriate primary antibodies overnight at 4°C. The antibodies used were anti-IKKα (sc-7218), anti-IKKβ (sc-34673), anti-NF-κB/p65 (sc-8008), anti-NF-κB/p50 (sc-8414), anti-IκBα (sc-1643) anti-p-IκBα (sc-8404), anti-Bax (sc-493), anti-Bcl2 (sc-7382), anti-COX-2 (sc-795), anti-cyclin D1 (sc-533), anti-PCNA (sc-56), and anti-VEGF (sc-152) procured from Santa Cruz Biotechnology, Santa Cruz, CA. Antibodies including anti-cleaved caspase-3 (#9661), anti-Bcl-xL (#2762), anti-p-IKKα/β (#2078) were purchased from Cell Signaling Technology, Danvers, MA. Anti-β-actin (#A1978) was obtained from Sigma-Aldrich, St. Louis, MO. The membrane was then incubated with appropriate secondary mouse (sc-2005) and rabbit (sc-2004) antibody horseradish peroxidase conjugate (Santa Cruz Biotechnology) followed by detection using chemiluminescence ECL kit (GE Healthcare Biosciences). For equal loading of proteins, the membrane was probed with appropriate loading controls.