Nitrocellulose nc membrane

Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM, Hyclone) supplemented with 1% penicillin/streptomycin (P/S) and 10% fetal bovine serum (Hyclone). Predesigned siRNA duplexes were purchased from Bioneer, and 10 nM siRNA transfection was carried out using RNAiMAX (Invitrogen, Waltham, MA, USA). Cells and tissues were lysed with N-PER™ and T-PER™ lysis buffer (Thermo Scientific, Waltham, MA, USA) supplemented with protease inhibitor cocktail (Roche, Hoffmann-La Roche AG, Basel, Switzerland), respectively. Lysates were resolved by SDS-PAGE gels and transferred onto nitrocellulose (NC) membranes (Merck Millipore, Burlington, MA, USA), and then antibodies were detected using the D-Plus ECL Femto System (Dongin Bio, Seoul, Korea) and the ImageQuant LAS4000 (GE Healthcare, Chicago, IL, USA). Antibodies used for immunoblotting included the following: anti-Ubiquitin (P4D1, Santa Cruz, Santa Cruz Biotechnology, Dallas, TX, USA), anti-UBE2H (18-Z, Santa Cruz), anti-Parkin (PRK8, Santa Cruz), anti-GAPDH (0411, Santa Cruz), anti-β-actin (C4, Santa Cruz), anti-Tau (Abcam, Cambridge, UK), and anti-UBE2L6 (MyBioSource, San Diego, CA, USA).

Cells were lysed by use of RIPA (Beyotime, Shangahi, China) containing protease inhibitor cocktail (Roche, Pleasanton, CA) and phenylmethylsulfonyl fluoride (Roche). Protein samples were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring onto nitrocellulose (NC) membranes (Sigma-Aldrich). After blocking via 5%-skim milk, the membranes were cultured with primary antibodies (dilution 1:1000) against Bax, Bcl-2, E-cadherin, N-cadherin, MRP2, MRP9, KNL1 (Cell Signaling Technology, Danvers, MA), followed by incubation with secondary antibodies (dilution 1:10000) for an hour at room temperature. β-actin or GAPDH was the loading control. Thereafter, signals were captured with the employment of the ECL chromogenic substrate.

CAP (99.0%), broflanilide (99.3%), cyclaniliprole (96.3%), flubendiamide (98.0%), tetraniliprole (97.5%), and benzamide (98.0%) were bought from the Shanghai Pesticide Research Institute Co., Ltd. (Shanghai, China). 3-Bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid (99.5%) and 2-amino-3-methyl-5-chlorobenzoic acid (98.0%) were purchased from the Shanghai Haohong Bio-pharmaceutical Science and Technology Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA), ovalbumin (OVA), N-hydroxysuccinimide (NHS), N,N-dimethylformamide (DMF), N,N’-dicyclohexylcarbodiimide (DCC), chloroauric acid (HAuCl₄), Freund’s incomplete adjuvant (FIA), and Freund’s complete adjuvant (FCA) were purchased from Sigma Aldrich (Shanghai, China). Tetramethyl benzidine (TMB) and polyethylene glycol-1500 (PEG1500) were purchased from Thermo-Scientific (Rockford, IL, USA). goat anti-mouse IgG horseradish peroxidase (HPR)-conjugated goat anti-mouse IgG and goat anti-mouse IgG were purchased from Boster (Pleasanton, CA, USA). Nitrocellulose (NC) membranes were purchased from Millipore (Boston, MA, USA). Microplates (96 and 24 wells) for cell culture and ELISAs were purchased from Conning Inc. (Corning, NY, USA). All organic reagents are of analytical purity, and biological reagents were prepared in double distilled water.

The AW ES antigens (20 μg) were subjected to sodium dodecyl sulfat-polyacrylamide gel (SDS-PAGE) in 5% stacking gels and 12% resolving gels at 80 V for 40 min and 120 V for 90 min. After electrophoresis, one gel was stained in Coomassie brilliant blue R-250 staining solution (Sigma, United States) for 4 h, and the other gel was used for the electrotransfer of proteins on nitrocellulose (NC) membranes (Millipore, United States) at 18 V for 35 min via a semi-dry transfer cell (Bio-Rad, United States) (Wang B. et al., 2013 (link)). Subsequently, the membranes were cut into strips, blocked with 5% skim milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.6) at 37°C for 1 h, and incubated at 4°C overnight with sera from different patients with trichinellosis at a dilution of 1:100. After being washed in TBST, the strips were incubated with HRP-conjugated goat anti-human IgG (1:5,000 dilutions; Sigma, United States) at 37°C for 1 h. Finally, the immunoreaction was detected with 3, 3-diaminobenzidine tetrahydrochloride (DAB; Sigma, United States). The gel and membranes were scanned using ImageScanner (GE healthcare, United States) and the MW of these bands was analyzed by AlphaView software (ProteinSimple, Santa Clara, CA, United States).

The standard method was used to analysis of the proteins extracted from FLSs by Western blot. The protein concentration was assayed by BCA Protein Assay Kit. The proteins were separated by 10% sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were electrophoretically transferred to nitrocellulose (NC) membranes (Millipore, Bedford, MA, United States). After blockage with 5% skim milk at room temperature for 2 h, the membranes were incubated with the specific primary antibodies against β-actin, RhoA, p38, p-p38, p65, p-p65, and F-actin at 4°C overnight on a rotary shaker. The membranes were washed in Tris-buffered saline/Tween 20 (TBST) and then incubated horse radish peroxidase conjugated secondary antibody for 2 h. The membranes were washed in TBST again and the immunoreactive proteins were detected with Pierce^TM ECL Western Blotting Substrate (Thermo Scientific, MA, United States). The protein bands were scanned by Alpha View SA system (ProteinSimple, San Jose, CA, United States) and analyzed using Image software.