Enhanced chemiluminescence reaction

Western blot analysis was performed as described previously. The podocytes, MPC5 cells were lysed in buffer containing 50 mmol/l Tris–HCl, pH 8.0, 150 mmol/l NaCl, 0.02% NaN₃, 0.1% SDS, 100 mg/l phenylmethylsulfonyl fluoride, 1 mg/l aprotinin, and 1% Triton. Cell extract was separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked for 1 h in TBST (10 mmol/l Tris–HCl, pH 7.4, 150 mmol/l NaCl, 0.05% Tween-20) containing 5% bovine serum albumin, incubated with primary antibodies Nephrin (Merck Millipore, Bedford, MA, USA), WT-1 (Merck Millipore), p-p38MAPK [Cell Signaling Technology (CST), Danvers, MA, USA], p-38MAPK (CST), p-p65 (CST), p-65 (CST), and GAPDH (CST) at 4°C overnight and then incubated with secondary antibodies. Bands were visualized with enhanced chemiluminescence reaction (Millipore Corp.). GAPDH was used as the loading control. Protein bands were captured and analyzed using the Lane 1D software (Sage Creation Science Co., Beijing).

For Western blot analysis (SDS–PAGE) total muscle lysates of EDL and SOL were prepared by adding 1/5 volume of fivefold concentrated electrophoresis sample buffer (310 mM Trise-HCl pH 6.8, 10 % SDS, 50 % glycerol, 100 mM DTT, 0.01 % bromophenol blue) to muscle lysates and boiled for 10 min at 80 ͦC. 30 μg of protein was separated by 7.5 % SDS–PAGE gel for immunological detection of selenoprotein N. Proteins were transferred electrophoretically to nitrocellulose membranes. After blocking in 5 % non-fat dry milk in PBS, membranes were incubated with primary antibodies overnight at 4 °C as follows: rabbit polyclonal anti-selenoprotein N antibody raised against amino acids 293–452 mapping within an internal region of selenoprotein N of human origin in 1:200, (Santa Cruz Biotechnology, INC, Santa Cruz, CA, USA); and rabbit polyclonal anti-actin antibody in 1:500 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After washing for 30 min in PBST, membranes were incubated with the HRP-conjugated secondary antibody (Bio-Rad, Hercules, CA, USA) in 1:1000 dilution in PBS containing 5 % non-fat dry milk for 1 h. Signals were detected by enhanced chemiluminescence reaction (Millipore, Billerica, MA, USA) according to the instructions of the manufacturer.

Total protein from different tissues or NECs was obtained following lysis using lysis buffer. Proteins in the lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, CA). After blocking with 5% nonfat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST), the membrane was incubated with the particular primary antibody, followed by horseradish peroxidase-conjugated secondary antibodies in blocking reagent. After extensive washing with TBST, immune reactive bands were analyzed by film exposure after enhanced chemiluminescence reaction (Millipore, Bedford, MA, USA).

Cells were lysed in buffer containing 50 mmol·L⁻¹ Tris/HCl, pH 8.0, 150 mmol·L⁻¹ NaCl, 0.02% NaN₃, 0.1% SDS, 100 mg·L⁻¹ phenylmethylsulfonylfluoride, 1 mg·L⁻¹ aprotinin and 1% Triton. Cell extract was separated by SDS/PAGE and transferred onto PVDF membranes. The membranes were blocked for 1 h in TBST (10 mmol·L⁻¹ Tris/HCl, pH 7.4, 150 mmol·L⁻¹ NaCl, 0.05% Tween‐20) containing 5% BSA, incubated with the primary antibodies anti‐E‐cadherin antibody (Proteintech), anti‐N‐cadherin antibody (Proteintech), anti‐vimentin antibody (Proteintech, Rosemont, IL, USA), anti‐α‐SMA antibody (Sigma), anti‐FAP antibody (Abcam), anti‐GDF10 antibody (R&D) at 4 °C overnight, followed by incubation with secondary antibodies. Bands were visualized with an enhanced chemiluminescence reaction (Millipore Corp., Billerica, MA, USA). GAPDH was used as the loading control. Protein bands were captured and analyzed using lane 1D software (Sage Creation Science Co., Beijing, China).

Total protein extracts were prepared using RIPA buffer (Sigma-Aldrich) supplemented with a protease inhibitor cocktail (Santa Cruz Biotechnology, Santa Cruz, CA). The protein concentration was determined by the bicinchoninic acid method, according to the manufacturer’s instructions (Pierce Chemical Co., Rockford, IL). Bovine serum albumin (Pierce Chemical Co.) was used as standard. Equal amounts of total proteins were separated on precast SDS-polyacrylamide gels (4–16%) (Bio-Rad, Marnes la Coquette, France), then transferred onto PVDF membranes (Bio-Rad). Membranes were incubated with mouse monoclonal anti-CYP1A1/2 (SC-53241, Santa Cruz) or goat polyclonal anti-actin (SC-1616, Santa Cruz) antibodies. Immunocomplexes were detected with horseradish peroxidase-conjugated mouse or goat secondary antibodies (Sigma) followed by enhanced chemiluminescence reaction (Millipore, Molsheim, France). Chemiluminescence was monitored using a ChemiDoc-XRS⁺ apparatus (Bio-Rad Laboratories) and quantified using the Image Lab software (version 4.1)

HSC3, OSCC3, and SCC4 cells were lysed in buffer containing 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.02% NaN₃, 0.1% SDS, 100 mg/L phenylmethylsulfonylfluoride, 1 mg/L aprotinin, and 1% Triton. Cell extracts were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked for 1 h in TBST (10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.05% Tween-20) containing 5% bovine serum albumin (BSA); incubated with primary antibodies against cleaved caspase-3 (Cell Signalling Technology (CST), Danvers, MA, USA), Bcl-2 (CST), PARP (CST), caspase-3 (CST), and GAPDH (CST) at 4 °C overnight; and then incubated with secondary antibodies. Bands were visualized with an enhanced chemiluminescence reaction (Millipore Corp.). GAPDH was used as the loading control. Protein bands were captured and analysed using LANE 1D software (Sage Creation Science Co, Beijing).

Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EDTA plus 10 µg/ mL aprotinin, 10 µg/ mL leupeptin, 1 mM PMSF, 1 mM Na₂VO₄) at +4 °C according to standard procedures. Twenty-five total protein extracts were resolved on 12% SDS/PAGE and electrically transferred onto poly(vinylidene difluoride) membranes (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked with TBS-T (20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.02% Tween-20) containing 5% skimmed milk (Bio-Rad Laboratories) for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies diluted in TBS-T containing 5% milk: rabbit polyclonal anti-Tom20 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) and mouse monoclonal anti-GAPDH (1:5000; Sigma-Aldrich). After thorough washing in TBS-T, immunocomplexes were revealed with horseradish-peroxidase-conjugated species-specific secondary antibodies (Jackson Laboratory, Bar Harbor, ME, USA) followed by an enhanced chemiluminescence reaction (Millipore Corporation, Darmstadt, Germany). Reactive bands were detected by the ChemiDoc MP system (Bio-Rad Laboratories) and signal quantification was performed using the IMAGE LAB software 5.0 (Bio-Rad Laboratories).