Autophagy Regulation in Grass Carp Cells

Grass carp LC3B was subcloned into pEGFP-N3 (Clontech, Mountain View, CA, USA) to generate GFP-LC3B to detect the formation of autophagy. Grass carp ATG5 was subcloned into pCMV-FLAG, pDsRed2-C1, and pEGFP-N3 (Clontech, Mountain View, CA, USA ) to generate FLAG-ATG5, DsRed-ATG5, and GFP-ATG5 to overexpress ATG5 in CIK cells. The plasmids were constructed as described previously [30 (link),31 (link)] Then, these vectors (500 ng/µL) were transfected into CIK cells by Lipofectamine™ 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s recommendations. Rapamycin, 3-MA, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG were purchased from Sigma (Saint Louis, MS, USA). Rabbit polyclonal anti-LC3B antibody was purchased from Abcam (Cambridge, UK). Rabbit polyclonal SQSTM1/P62 antibody was purchased from Beyotime (Shanghai, China). Lyso-Tracker Red fluorescent probe was purchased from Solarbio (Beijing, China).