Polyvinylidene difluoride membrane

The total protein was isolated from liver tissues using RIPA buffer (Sigma-Aldrich, St. Louis, US). Ten micrograms of total protein was loaded per well and then separated on 12% sodium dodecyl sulfate-polyacrylamide gel. Then, the protein was transferred onto the polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, United Kingdom). The membrane was blocked with 5% BSA in TBST (0.5% Tween 20) for 1 h at room temperature. The membrane was incubated at 4°C overnight with primary antibodies: rabbit anti-phospho-AKT (Ser473, 1 : 1000), rabbit anti-phospho-Stat3 (Tyr705, 1 : 2000), rabbit anti-phospho-NF-κB p65 (Ser536, 1 : 1000), rabbit anti-AKT (1 : 1000), rabbit anti-Stat3 (1 : 2000), anti-NF-κB p65 (1 : 1000), rabbit anti-caspase 3 (1 : 1000), and rabbit anti-GAPDH (1 : 20000, Sigma-Aldrich, St. Louis, USA). All antibodies besides rabbit anti-GAPDH were purchased from Cell Signaling Technology, Beverly, USA. The membrane was incubated for 1 h at room temperature with a goat polyclonal to rabbit IgG antibody (1 : 10000, Abcam) and developed with the SuperSignal West Femto Trial kit (Thermo, USA). The membrane was exposed to high-sensitivity films (GE Healthcare) for autoradiography.

The expression levels of AR and p53 were examined by Western blotting. Proteins were extracted and quantified using Nanodrop 1000 (Thermo Fisher Scientific). Equal amounts of proteins were separated in10% SDS-PAGE gel (Invitrogen) and blotted onto the polyvinylidene difluoride membrane (GE Healthcare Life Sciences). Membranes were probed with antibodies against the proteins [AR (Cell Signaling Technology, catalog no. 3202, RRID:AB_2060162) and p53 (Santa Cruz Biotechnology, catalog no. sc-126, RRID:AB_628082)] and β-actin (Sigma-Aldrich, catalog no. A3854, RRID:AB_262011) as control. Membranes were incubated with second antibodies, followed by detection of ECL reagent (GE Healthcare Life Sciences), and the Amersham Imager 600 (GE Healthcare Life Science).

To extract total protein from lenses, lenses were homogenized in lysis buffer composed of protease and phosphatase inhibitor cocktails for 1 h at 4 °C. The extracted protein concentration was quantified by BCA protein assay (Pierce), subjected to 7.5–10% SDS-Tris glycine gel electrophoresis, transferred to polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK). The membrane was incubated in 5% non-fat skim milk in with Tween-20 buffer (10 mmol/L Tris, 150 mmol/L NaCl, and 0.1% Tween-20, pH 7.4) for 1 h. Primary antibodies: AKR1B1, MMP9, vimentin (GTX113381, GTX100458 and GTX100619, respectively, Genetex, Irvine, CA, USA), N- and E-cadherin (610920 and 610182, respectively, BD Biosciences, Franklin Lakes, NJ, USA), Phosph-AMPK α1,2^T172 (44-1150G, Thermo Fisher Scientific, Eugene, OR, USA), and SOD2 (acetyl K68) (ab137037, Abcam, Eugene, OR, USA) were diluted in PBST at 1:2000 dilutions and incubated at 4 °C overnight. Peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:10,000) were used. The result was visualized using ECL-Plus detection kit (GE Healthcare) and exposed to film. The developed films were scanned (photo scanner 4490, Epson, Long Beach, CA, USA), analyzed using NIH image densitometry analysis software (National Institutes of Health, Bethesda, MD, USA).

The phosphorylation of ERK1/2 and JNK, and the expression of activated caspase‐3, PARP‐1, SIRT1, RANKL, sclerostin and Ac‐p53 were performed by western blot in MLO‐Y4 cells seeded in 60 mm tissue culture dish treated as reported above. Cells were lysed for 30 min at 4 °C in ice‐cold RIPA buffer and centrifuged at 20 000 g for 10 min. Equal amounts of total proteins (40–60 μg) were loaded in each line and were subjected to SDS/PAGE on 10% gel and electrotransferred to poly(vinylidene difluoride) membrane (GE Healthcare). Membranes were probed with specific primary antibody anti‐caspase‐3 or anti‐phospho‐ERK1/2 or anti‐phospho‐JNK or anti‐PARP‐1 (Cell Signalling Technology, Danvers, MA, USA) or anti‐SIRT1 or anti‐RANKL or anti‐sclerostin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), or anti‐Ac‐p53 (Thermo Fisher Scientific). Subsequently, after stripping the membranes were reprobed with anti‐ERK1/2 or anti‐JNK or anti‐β‐actin for normalization of densitometric values. Secondary antibodies conjugated to horseradish peroxidase were used to detect antigen–antibody complexes using a chemiluminescence reagent kit (Clarity Western ECL Substrate, Bio‐Rad, Hercules, CA, USA). imagej software (National Institutes of Health, Bethesda, MD, USA) was used to perform quantitative analyses, and the values of the bands were expressed as fold‐increase relative to control values.

Proteins were transferred from the gel to a polyvinylidene difluoride membrane (GE Healthcare) which was blocked with 5% milk powder in TBS-T (Sigma-Aldrich) for 60 min. The membrane was incubated with a primary antibody overnight, followed by incubation with the peroxidase conjugated secondary antibody for 60 min. Blots were visualized by chemiluminescence (SuperSignal West Dura Chemiluminescent Substrate, Pierce) and documented using the ChemiDoc system (Bio-Rad).