Ecl system

The tumor and control tissues are lysed with cell lysis buffer with 1 mM phenylmethanesulfonyl fluoride (PMSF) and 1×protease inhibitor cocktail (Roche). For Western blot analysis, the protein sample concentration was adjusted to 40 μg/μl, separated by SDS-PAGE, then switched to a PVDF membrane, and incubated with the corresponding primary antibody at 4°C overnight. The next day, the corresponding secondary antibody was incubated for 1 h at room temperature. An ECL system (Cell Signaling Technology) was used to detect protein signals. The GAPDH protein in the whole protein was used as an internal control.

Dissected E18.5 chicken tissues or cultured cells were homogenized in ice-cold lysis buffer consisting of 150 mM NaCl, 50 mM Tris at pH 7.5, 1% (vol/vol) Triton X-100, 1 mM PMSF, and 1× protease inhibitor cocktail (Roche). After centrifugation at 4 °C, the supernatant was collected and separated by polyacrylamide gel electrophoresis (PAGE), then transferred to PVDF membrane and incubated with corresponding primary antibodies, followed by incubation with secondary antibodies (Bio-Rad). The signals were detected with the ECL system (Cell Signaling Technology).

Mouse tissues were homogenized in ice-cold lysis buffer consisting of 150 mM NaCl, 50 mM Tris at pH 7.5, 1% NP-40, 0.1% SDS, 1% (vol/vol) Triton X-100, 1 mM PMSF, and 1 × protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO, United States). The supernatant was collected after centrifugation at 4°C and separated by polyacrylamide gel electrophoresis (PAGE), then transferred to PVDF membrane. After blocking in PBS containing 5% milk and 0.1% Tween-20, the membrane was incubated with primary antibody at 4°C over night, followed by incubation with HRP-conjugated secondary antibody (Bio-Rad, Cat. No. 170-6515 or 170-6516) at room temperature for an hour. The signals were detected with the ECL system (Cell Signaling Technology, Danvers, MA, United States). Primary antibodies used are as follows: anti-ERM (rabbit, Cell Signaling Technology, Cat. No. 3142), anti-pERM (rabbit, Cell Signaling Technology, Cat. No. 3726), anti-CDK5 (rabbit, Santa Cruz, Cat. No. sc-173), and anti-GAPDH (mouse, Millipore, Cat. No. MAB374).

Proteins for Western blotting were isolated by lysing cells in lysis buffer (50 mM Tris pH 7.4, 0.8 M NaCl, 5 mM MgCl2, 0.1% Triton X-100) containing protease inhibitor and a phosphatase inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), incubated on ice for 15 min and cleared by microcentrifugation. Twenty micrograms of total protein/lane were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a PVDF membrane. The membranes were incubated overnight at 4 °C with primary antibodies. After washing in T-TBS, the membranes were incubated with peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000) for 2 h at room temperature. The immune complex was visualized with an ECL system (Cell Signaling Technology, Danvers, MA, USA). Protein expression levels were quantified using Image J (National Institutes of Health, Bethesda, MD USA) and were expressed as fold changes relative to the control treatment. The primary antibodies (anti-p-AMPKα1-thr172, p-ACC-ser79 and pLKB1-ser428) and the antibodies against the corresponding total forms were obtained from Cell Signaling Technology (Danvers, MA, USA). TRPV1 was obtained from Thermo Scientific (Waltham, MA, USA). Peroxidase-labeled secondary anti-mouse IgG was from Sigma-Aldrich (St. Louis, MO, USA) and anti-rabbit IgG was from Cell Signaling Technology (Danvers, MA, USA).

COS7 cells were transfected with expression vectors in 6-well plates. Forty-eight hours after transfection, cells were washed with PBS and lysed in ice-cold lysis buffer containing 150 mM NaCl, 50 mM Tris at pH 7.5, 1% Triton X-100, and 1 mM PMSF. After centrifugation at 4°C, the supernatant was collected and separated by 10% polyacrylamide gel electrophoresis (PAGE), then transferred to PVDF membrane. The membrane was blocked in 5% nonfat milk for an hour at room temperature, then incubated with anti-GFP antibody (ABclonal, AE012) at 4°C overnight, followed by incubation with HRP-conjugated secondary antibody (Bio-Rad, 170-6516) for an hour at room temperature. The signals were detected with the ECL system (Cell Signaling Technology).

The protein of human LECs, cortex, and rat lens in each group were extracted in RIPA lysis buffer III (Sangon Biotech, China) as per the instructions. After ultrasonication, the specimens were centrifuged at 12000 rpm (15 min, 4°C), and the supernatant was collected. Equal amounts of lysates (100 μg/lane) were separated on 10% SDS-PAGE and transferred to PVDF membranes. After blocking in 5% skimmed milk (TBST as vehicle) for 2 h, the membranes were incubated with rabbit anti-LSS antibody (1 : 500, Abcam, UK) and mouse anti-GAPDH antibody (1 : 5000, ABclonal, France) overnight at 4°C. After washing, the membranes were incubated with goat HRP-conjugated secondary antibodies (Jackson ImmunoResearch, USA). Proteins were visualized using an ECL system (Cell Signaling, USA). Relative level of proteins was quantified with ImageJ software (National Institutes of Health, USA).

Cells were harvested and lysed in cell lysis buffer (Cell Signaling Technology, MA). The proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Roche, Basel, Switzerland). The membranes were incubated with primary antibodies against HIF-1α, phospho-histone H2AX (Ser139), and GAPDH overnight at 4 °C. After incubation with HRP-conjugated secondary antibody for 1 h at room temperature, bands were detected using an enhanced chemiluminescence (ECL) system (Cell Signaling Technology). GAPDH served as an internal reference.