Anti irak1

Cells were suspended in Radio Immunoprecipitation Assay lysis buffer (Thermo Scientific, PA, USA) supplemented with protease inhibitor (Sigma Aldrich). Cytosol and nuclear fractions were isolated according to manufacturer’s instruction (Thermo). Equivalent proteins were subjected to SDS-PAGE and transferred to polyvinyl difluoride membranes (Millipore Corp., Atlanta, GA, USA) for western blotting. After blocking membrane with 5% non-fat dry milk for 2 h, we incubated it with primary antibodies at 4°C overnight. Primary antibodies included anti-FLAG M2 (Sigma), anti-Omp25, anti-Omp31 and anti-β-actin (Wuhan boster Biotech), anti-phospho-IκB, anti-IκB, anti-p65, anti-TRAF6, anti-IRAK1, anti-IRAK2, and anti-Histone H3 (Cell Signaling Technology). HRP-conjugated secondary antibodies were incubated at room temperature for 1 h. ECL (Bio-Rad) was used for enhanced chemiluminescence detection according to the manufacturer’s instructions.

Total protein of cells was harvested in RIPA lysis buffer (Sigma-Aldrich, MO, USA) containing a protease inhibitor. The protein concentration of the whole-cell lysate was determined by the BCA Protein Assay (Sigma-Aldrich, MO, USA). Then, protein samples were separated by standard 10% SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). The membranes were then blocked in 5% fat-free milk for 1 h at room temperature, dissolved with Tris-buffered saline-Tween (TBST). Then, all membranes were incubated overnight at 4°C with the primary antibodies: anti-IRAK1 (#4504; Cell Signaling Technology, MA, USA; 1 : 1000), anti-E-cadherin (20874-1-AP, Proteintech, 1 : 5000), anti-N-cadherin (22018-1-AP, Proteintech, 1 : 2000), anti-Vimentin (10366-1-AP, Proteintech, 1 : 1000), and anti-GAPDH (10494-1-AP, Proteintech, 1 : 5000). After washing with TBST three times, the membranes were then incubated with the secondary antibody for 1.5 h at room temperature. The bands were finally visualized using an ECL reagent (Millipore, MA, USA) after washing four times with TBST solution.

Cells were harvested in lysis solution containing 50 mM Tris/HCl (pH 7.6), 1 % NP40, 150 mM NaCl, 2 mM EDTA, 100 μM PMSF, a protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland), and a phosphatase inhibitor (Sigma-Aldrich). After incubation on ice for 30 min, cellular debris was removed by centrifugation (10 min, 4 °C). Proteins (10 μg) were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane. The following antibodies were used: anti-β-actin (Sigma-Aldrich), anti-TG2 (Neomarkers, Fremont, CA), anti-E-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA), N-Cadherin (Santa Cruz Biotechnology), anti-phospho-NF-kB p65 (S276) (Cell Signaling, Danvers, MA), anti- NF-kB p65 (Cell Signaling), anti-I-kB (Santa Cruz Biotechnology), anti-phospho-JNK (Santa Cruz Biotechnology), anti-JNK (Santa Cruz Biotechnology), anti-IRAK1 (Cell Signaling), anti-IRAK2 (Cell Signaling), and anti-TRAF6 (Santa Cruz Biotechnology).

To prepare whole-cell extracts, MG6 cells were lysed in cold buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 0.1% SDS, and 1% NP-40. The lysis buffer was supplemented with proteinase and phosphatase inhibitors (Roche Applied Science, Mannheim, Germany). Thereafter, the protein amount was determined using the Lowry method and denatured in SDS gel loading buffer. Equal amounts of proteins were separated using 10–20% SDS-polyacrylamide gel (Wako Pure Chemicals) and transferred via a semi-dry transfer cell (Bio-Rad) into a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with TBS-T containing 3% bovine serum albumin (Sigma-Aldrich), the membranes were incubated overnight at 4 °C with the following primary antibodies: anti-phospho-NF-κB p65 (Ser536), anti-NF-κB p65, anti-phospho-IκBα (Ser32), anti-IκBα, anti-phospho-IKKα/β (Ser176/180), anti-IKKβ, anti-phospho-TAK1 (Thr184/187), anti-TAK1, and anti-IRAK1 all from Cell Signaling Technology (Danvers, MA, USA), as well as anti-TRAF6 from Funakoshi (Tokyo, Japan). Furthermore, the protein bands were visualized using Immobilon Western Detection Reagent (Millipore) using the ChemiDoc Imaging System (Bio-Rad, Richmond, CA, USA), and densitometric analysis was performed using Image Lab 6.1 (Bio-Rad) normalized to α-tubulin (Sigma-Aldrich) or anti-lamin (Cell Signaling Technology) as the loading control.

Cell lysates were prepared with RIPA buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, pH 7.5, 0.1% SDS and 0.5% sodium deoxycholate supplemented with 1X protease inhibitor cocktail) for Western blotting. Protein concentrations were determined by BCA protein assay kit (Novagen). Protein samples were run on 12% SDS gel and transfer was performed at 100 V for 2 hour onto PVDF membrane (Millipore). Membranes were blocked in 5% skimmed milk. Membranes were incubated with primary antibody at 4°C for overnight. Membranes were washed with three washes of 15 min each with TBST buffer. HRP conjugated secondary antibody was incubated onto membrane for 1 hour and followed by three washes for 15 min each with TBST buffer. The membranes were developed by using Super-Signal developing reagent (Pierce). Antibodies, anti-TRAF6, anti-IRAK1, anti-IRAK2, anti-phospho NF-κB p65, anti-NF-κB p65 (Cell Signaling Technology) and anti-β-tubulin (Abcam) have been used in the study. Western blots band intensities were quantified by using ImageJ software and normalized by β-tubulin image density.