Phenylmethylsulfonyl fluoride

PC12 cells were collected and treated with RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing 1% (v/v) phenylmethylsulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.), 0.3% (v/v) protease inhibitor (Sigma-Aldrich; Merck KGaA) and 0.1% (v/v) phosphorylated proteinase inhibitor (Sigma-Aldrich; Merck KGaA). Western blots were performed as previously described (22 (link)). Membranes were incubated with primary antibodies against TRPC6 (cat. no. ab62461; 1:1,000; Abcam) and anti-GAPDH (cat. no. 2118; 1:5,000; Cell Signaling Technology, Inc.) overnight at 4˚C. Membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (cat. no. ZB-2301; 1:5,000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) for 2 h at room temperature, followed by three washes with TBS-Tween-20. Enhanced chemiluminescence (ECL; EMD Millipore) was used to determine protein concentrations according to the manufacturer's protocol. Protein signal was detected using a Super ECL Plus kit (Nanjing KeyGen Biotech Co., Ltd.). Relative protein expression was normalized to GAPDH. All experiments were repeated three times. ImageJ v1.43b software (National Institutes of Health) was used for densitometric analysis.

T24 and 5637 cells were cultured in six-well plates and exposed to various concentrations of MLN4924 (0, 0.1, 0.3, 1 μM), with or without celecoxib (60 μM) for 24 h. Then, the cells were harvested at 80% confluency with a cell scraper and lysed in RIPA lysis buffer (Solarbio) with 1-mM phenylmethylsulfonyl fluoride (PMSF) and 1%-mM phosphatase inhibitors (Solarbio) on ice for 30 min. After measuring protein concentration by the Pierce BCA protein Assay kit (Thermo, USA), the samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). After blocking with 5% skim milk for 1 h (Solarbio), the membranes were incubated with primary antibodies AKT, phospho-AKT (S473), p44/42MAPK (ERK1/2), phospho-p44/42MAPK (ERK1/2), PARP, Bcl2, BAX, N-cadherin, E-cadherin, vimentin, and β-actin (CST, Danvers, MA, USA) at 4°C overnight. Then, the membranes were washed with tris buffered saline tween (TBST, Solarbio) three times (10 min each) and incubated with horseradish peroxidase-conjugated secondary antibodies to IgG (CST) for 1 h at room temperature. The PVDF membrane was quantified using LAS-4000 Mini (Fuji Film, Japan).

Animals were deeply anesthetized with 1% pentobarbital sodium (0.1 g/kg, i.p.) and the ipsilateral spinal cord in the lumbar enlargement site was rapidly separated and homogenized in ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride and protease phosphatase inhibitor (Solarbio Company, China). After being kept on ice for 1 h, the lysates were centrifuged at 12,000 rpm for 15 min at 4 °C and the supernatants mixing with loading buffer were denatured at 95 °C for 5 min. The protein concentrations were measured using a bicinchoninic acid (BCA) kit (Pierce Biotechnology Inc.).