Goat anti mouse igg hrp

The cell total protein was isolated using RIPA (Applygen Technologies Inc., Beijing, China), and protease inhibitor (CWBIO, Shanghai, China) was added into the RIPA at a ratio of 1:100. After adding RIPA to the cell culture plate, we collected the cells, then followed this by centrifuging (12,000 rpm) at 4 °C for 10 min. Protein concentrations were measured using the Thermo Scientific Pierce BCA protein assay kit (Thermo Fisher, USA), and 1/4 volume of 5× loading buffer was added to the supernatant. A total of 20 µL of protein was blotted using 10% SDS-polyacrylamide gel, and transferred to a polyethylene difluoride (PVDF) membrane (CST, Boston, MA, USA). After blocking with 5% defatted milk for 2 h, membranes were incubated with antibodies (1:1000) against Gli1 (BOSTER, Wuhan, China), Gli2 (Wanleibio, Shenyang, China), Gli3 (Wanleibio, China), PGC1α (Abcam, Cambridge, MA, USA), PPARγ (Abcam, USA), UCP1 (Abcam, USA), aP2 (Abcam, USA), Cox7a (Abcam, USA), and β-tubulin (Abcam, USA) overnight at 4 °C. The membrane HRP goat anti-mouse IgG, goat anti-rabbit IgG, or rabbit anti-goat IgG secondary antibodies (BOSTER, China) were diluted 1:3000 and incubated for 1 h. Detection was performed using chemiluminescence western blotting substrate (Santa Cruz, CA, USA), with lmage Lab analysis software (Image Lab™, Bio-Rad, Berkeley, CA, USA).

After H/R irritation and/or OE-Myo1b (or si-Myo1b) transfection, total proteins of H9c2 cells were detached using RIPA Lysis Buffer (Beyotime Biotechnology). BCA assay (Beyotime Biotechnology) was performed to measure protein concentration. Then, proteins in equal concentration were electrophoresed on polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membrane, which were incubated with anti-Myo1b antibody (ab194356, 1 : 1000, 60 min), anti-Bcl-2 antibody (ab196495, 1 : 1000, 40 min), anti-Bax antibody (ab32503, 1 : 1000, 40 min), anti-LC3B antibody (ab63817, 1 : 500, 40 min), anti-p62 antibody (ab91526, 1 : 1000, 60 min), anti-GAPDH antibody (ab8245, 1 : 10000, 40 min, Abcam Biotechnology, CA, USA), or anti-cleaved-caspase 3 antibody (#9661, 1 : 1000, 60 min, Cell Signaling Technology, MA, USA). Then, PVDF membranes were incubated with HRP goat anti-mouse IgG (BA1051, 1 : 10000) or HRP goat anti-rabbit IgG (BA1054, 1 : 10000, Boster Biological Technology, Wuhan, China) for 60 min. Results were visualized via enhanced chemiluminescence technique. Intensities of proteins were analyzed via the Image-Pro Plus 6.0 software.

Cell samples were lysed in RIPA buffer (Beyotime, Co., Ltd., People’s Republic of China) to obtain total cell lysates. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Co., Ltd., U.S.A.). Protein (40 μg) from each sample was taken out to perform SDS/PAGE, and then transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany) and further incubated with primary polycloncal antibodies: LHFPL3 (1:5000), E-cadherin (1:1000), N-cadherin (1:1500), vimentin (1:1000), snail (1:2000), followed by incubation with horseradish peroxidase conjugated monoclonal HRP goat anti-rabbit (BOSTER, No.BA1054, 1:20,000) and HRP Goat anti-Mouse IgG (BOSTER, No.BA1051,1:20,000) antibodies. The membranes were stripped and reprobed with a primary monoclonal mouse anti-rabbit antibody against GAPDH (1:1000 dillution; Bioworld, Nanjing, People’s Republic of China). GAPDH expression was used as a standard for the normalization of the measurement.