Anti lc3 antibody

All biochemicals, cell culture reagents, Dulbecco Modified Eagle Medium (DMEM), fetal bovine serum (FBS), protease inhibitor cocktail, bovine serum albumin (BSA) selective S1P₂ antagonist JTE013, specific S1P_1/3 antagonist VPC23019, specific inhibitor of SK1 PF-543, and myosin heavy chain (MHC) antibody were purchased from Merck Life Science (Burlington, MA, USA). Selective S1P₁ antagonist W146 was from Avanti Polar Lipids (Alabaster, AL, USA). Recombinant TNFα was obtained from PeproTech (London, UK). Human specific TaqMan Gene Expression Assays employed for gene expression studies were purchased from Thermo Fisher Scientific INC (Waltham, MA, USA). Anti-SK1, anti-SK2, anti-phospho-SK1 (Ser225) and anti-phospho-SK2 (Thr578) antibodies were from ECM Biosciences (Versailles, KY, USA). Secondary antibodies conjugated to horseradish peroxidase, anti-LC3 antibody and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The cells were seeded in 20 mm glass-bottom cell culture dishes. After treatment, the cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 for 20 min. Then, cells were blocked with 3% bovine serum albumin (BSA) for 30 min at room temperature and incubated with anti-LC3 antibody (1 : 100, Santa Cruz Biotechnology, Inc., USA) overnight at 4°C. The cells were washed twice with PBS and incubated with the corresponding secondary antibody for 1 h at 37°C. Nuclei were stained with DAPI for 5 min at 37°C in the dark. After the cells were washed twice with PBS, they were examined by laser-scanning confocal microscopy (LSCM; LSCM 510 Meta; Zeiss, Gottingen, Germany).