Western blot
Standard western blotting was conducted for protein expression assays in
hypoxic H9C2 cells transfected with miR-210 mimic, inhibitor and NC. Two
days following transfection, proteins were isolated with RIPA lysis
buffer containing 1 mg protease inhibitors (Applygen Technologies Inc.,
Beijing, China). The protein contents were quantified using the
Bicinchoninic Acid (BCA) Protein Assay Kit (CoWin Biotech Co., Ltd.,
Beijing, China). Primary antibodies against light chain 3
(LC3;
ab48394), p62 (ab56416), Caspase8ap2
(ab4052), cleaved caspase 8 (ab25901), cleaved caspase 3 (ab13847),
Beclin-1 (ab62557), and the internal controls β-actin and GAPDH were
from Abcam (Cambridge, UK). Furthermore, secondary antibodies labelled
with horseradish peroxidase were incubated with membranes for 1 h at
37°C.
Samples
were then transferred to a polyvinylidene fluoride (PVDF) microporous
membrane (Millipore, Boston, USA). Blots were imaged by a FluorChem E
imager (Cell Biosciences, San Jose, USA). The grey value of each band
was determined with ImageJ version 1.51.