Anti atg5 antibody

For western blotting, total protein was extracted from cardiac tissue and myocyte cells using a RIPA buffer and separated by SDS-PAGE, and the protein concentration was examined with a BCA protein assay kit (KGP902; Keygen, China). The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and blocked with 5% nonfat milk powder for 1 h. The membranes were probed with the following primary antibodies: anti-IKKε antibody (1 : 1000; Cell Signaling, #2690); anti-LC3 antibody (1 : 2000; Abcam, USA, #ab51520); anti-ATG-5 antibody (1 : 1000; Cell Signaling, USA, #2630); anti-p-AKT antibody (1 : 1000; Cell Signaling, USA, #S473); anti-AKT antibody (1 : 1000; Cell Signaling, USA, #C67E7); anti-mTOR antibody (1 : 1000, Cell Signaling, USA, #7C10); and anti-p-mTOR antibody (1 : 1000; Cell Signaling, USA, #S2448). The following day, the PVDF membranes were incubated with secondary antibodies (1 : 5000; Cell Signaling) at room temperature for 1 h. Specific proteins were detected using an ECL reagent (GE Healthcare, Piscataway, NJ, USA) and captured on Hyperfilm (Amersham, GE Healthcare). The results were then analyzed using the ImageJ software for semiquantitation of the mean gray value of each blot.

The pcDNA EGFP-RFP-LC3B plasmid encoding EGFP, RFP, and LC3B was constructed by the pcDNA 3.1 myc-his (-) vector. The pcDNA EGFP-mRFP-LC3B (ptfLC3) plasmid encoding Chlorocebus sabaeus LC3B (NCBI Reference Sequence: XM_007994295.2) was constructed by the pcDNA 3.1 myc-his (-) vector. Anti-LC3B antibody (Cell Signaling, Danvers, MA, USA, #2775), anti-SQSTM1/p62 antibody (Cell Signaling, #5114), anti-ATG5 antibody (Cell Signaling, #9980), anti-ATG12 antibody (Cell signaling, #4180), anti-LAMP1 (Cell Signaling, #3243), anti-β-actin (Santa Cruz, sc-47778), and mouse anti-PEDV antibody were made in our laboratory (immunized inactivated PEDV in mouse).