Horseradish peroxidase linked secondary antibody

The mitochondrial fraction from the heart muscle was carried out according to the method described by Pecqueur et al. [33 (link)]. Briefly, the tissue was homogenized at 4 °C in a TES buffer (10 mM Tris pH 7.5, 1 mM EDTA, 250 mM sucrose and 5 μL/mL protease inhibitor cocktail (Sigma)). After centrifugation, the mitochondrial pellet was resuspended in TES buffer and stored at −80 °C. The protein content was quantified by the Bradford assay. Protein samples (50 μg protein/lane) were resolved by SDS-PAGE and transferred to PVDF membranes. The membranes were probed with a specific antibody (goat polyclonal anti-UCP2 from Santa Cruz Biotechnology, Inc.). The blot was incubated with horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology) followed by chemiluminescence detection as described above. The protein levels were normalized to actin.

Frozen heart powder was homogenized in a lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100 and 5 μL/mL protease inhibitor cocktail (Sigma)) as described by Bogazzi et al. [32 (link)]. After 30 min incubation on ice, the lysates were centrifuged at 4 °C and supernatants were stored at −80 °C. The protein content was quantified by the Bradford assay. Total protein samples (100 μg/lane) were resolved on SDS-PAGE, transferred to PVDF membranes and probed with specific antibodies (rabbit polyclonal anti M-CPT1 or rabbit polyclonal anti-PPARα from Santa Cruz Biotechnology, Inc.). The blots were incubated with horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology) followed by chemiluminescence detection as described above. The protein levels were normalized to actin.

Western blotting was performed according to the standard procedures. An equal amount of each protein lysate was loaded onto 8% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and blotted onto polyvinylidene fluoride membranes. Samples were blocked in Tris-buffered saline Tween 20 (20 mM Tris-HCl, pH 7.4 and 0.05% Tween 20) with 5% non-fat dry milk. The membranes were incubated with primary antibodies at appropriate dilutions overnight at 4°C and horseradish peroxidase-linked secondary antibody (at a dilution of 1:2000) (Santa Cruz, USA) for 1 hour at room temperature. The results were visualized by fluorography using the Tanon gel imaging system (Tanon, Shanghai, China).

Western blotting was performed as previously described with minor modifications^{14 (link)}. Total protein was extracted and then separated by the use of 12% SDS-polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene difluoride membrane. The membrane was placed in a 5% fat-free skimmed milk solution in Tris-buffered saline/0.05% Tween 20 for at least 1 h at room temperature, followed by an anti-Gal1 antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were stripped and re-probed with an anti-β-actin antibody (Santa Cruz Biotechnology) to validate equalized lane loading. Membranes were treated with a horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. The resulting bands were visualized using an enhanced chemiluminescence system (Pierce Biotechnology, Rockford, IL, USA). Experiments were performed in triplicate.

The cells were seeded at 3 × 10⁵ cells per well in six-well plates for 24 h, and then treated with LDM as indicated. The cells were washed with iced PBS and lysed with lysis buffer containing protease inhibitors cocktail (Cell Signaling Technology) plus 1 mM PMSF. Protein concentration was determined by Bradford assay. In all, 50 μg of protein extracts were loaded and resolved by SDS-polyacrylamide gel electrophoresis, and transferred to PVDF membrane (Millipore Corporation, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk PBS-T buffer at room temperature for 1 h, and then incubated for 2 h with primary antibodies at 1 : 1000 dilutions except for β-actin (1 : 5000). The membranes were then incubated for 1 h with an appropriate horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and electrochemiluminescence was performed with ChemiImager 5500 imaging system (Alpha Innotech Corporation, San Leandro, CA, USA). Intensity values of representative blots were determined with ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to the intensity of respective loading control β-actin in each lane.

For immunoblotting, cells were lysed in a solution containing 50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, complete protease inhibitor (Roche) and phosphatase inhibitors I & II (Sigma) and analysed by SDS-PAGE using 4 to 12% Bis-Tris gels (NuPAGE; Invitrogen), followed by electrotransfer onto Invitrolon polyvinylidene difluoride membranes (Invitrogen). Membranes were blocked with 5% (wt/vol) non-fat dry milk and 0.1% (vol/vol) Tween-20 in Tris-buffered saline for 1 hour at room temperature, before being probed with the primary antibody by overnight incubation at 4 °C, followed by incubation for 1 hour at room temperature with a horseradish peroxidase-linked secondary antibody (Santa-Cruz) and detection using ECL reagents (Bio-Rad, Hercules, USA), following the manufacturer’s protocol. Immunoblots were quantified by scanning of films with a calibration strip and analysis by densitometry using Image J (US National Institutes of Health; http://rsb.info.nih.gov/ij).

Cells or tissue specimens were homogenized in RIPA lysis and extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA), separating lysates by electrophoresis in 12% sodium dodecyl sulfate-polyacrylamide gel. The various proteins were then electrophoretically transferred onto polyvinylidene fluoride membranes for blocking (5% skim milk, 1 h) and overnight incubation (4 °C) with the following primary antibodies: K1 (Abcam), K10 (Abcam), K14 (Abcam), Ki-67 (Acris Antibodies), p-AKT (Cell Signaling Technology, Danvers, MA, USA), AKT (Cell Signaling Technology), involucrin, and β-actin (Santa Cruz Biotechnology). Immunoreactive bands were developed using a horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology) and visualized as directed by light emission (Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate; PerkinElmer, Waltham, MA, USA). Quantitative assessment of immunoreactivity was software-enabled (IMT Inc).