Anti rabbit polyclonal atg8 lc3 antibody

The tissue samples from the frozen specimens were dissected and homogenized in lysis buffer with a Dounce homogenizer. Samples were lysed using radioimmunoprecipitation assay buffer with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The lysed tissues were centrifuged at 10,000 × g for 20 minutes for clarification. After quantification of the protein content, the lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12%-15% acrylamide gels. Immunoblotting was conducted after transferring the lysate to polyvinylidene difluoride membranes (Millipore, Billerica, MA). The corresponding antibodies were as follows: anti-rabbit polyclonal atg8/LC3 antibody, SQSTM1/p62 antibody, and Beclin 1 antibody, obtained from Cell Signaling (Irvine, CA); phospho-JNK, p38, and p65, obtained from Cell Signaling (Danvers, MA); interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and β-actin (sc-70,319), purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Chemiluminescence Western Blotting Detection Reagents (Millipore) were used for the detection of bands, and the quantification of blots was conducted with ImageJ densitometry software (National Institutes of Health, Bethesda, MD) for densitometric analyses.