Rabbit anti lc3a b antibody

Western blotting analysis was performed as described previously [22 (link)]. Antibodies used in this experiment were as follows: goat anti-COTE1 (1 : 200; Santa Cruz Biotechnology, CA, USA), rabbit anti-LC3A/B antibody (1 : 1000; Cell Signaling Technology, Massachusetts, USA), mouse anti-P62 antibody (1 : 1000; Cell Signaling Technology, Massachusetts, USA), rabbit anti-Beclin-1 antibody (1 : 200; Santa Cruz Biotechnology, CA, USA), and anti-β-actin (1 : 500; Santa Cruz Biotechnology, CA, USA). The gray value of proteins was quantitatively analyzed by ImageJ (v.1.8.0).

For protein extraction, 50 μl of laemmli buffer (0.05% (w/v) bromophenol blue, glycerol (30% (v/v)), 5 M EDTA, NaOH solution, 6% (w/v) SDS and 1.875 M Tris pH 8.8 solution) and 1% (v/v) Dithiothreitol, (all from Sigma-Aldrich, USA) were used. Samples were macerated and denatured for 20 min at 45 °C followed by 20 min 65 °C and finally 10 min at 90 °C. For the immunoblotting assay, 10 μg of total protein extract were resolved in a 12% SDS gel and transferred to a nitrocellulose membrane for 10 min in Trans-Blot Turbo transfer system (Bio-Rad, USA). Membranes were blocked in Tris-buffered saline (TBS) with 0.1% tween 20 (TBS-T) containing 5% BSA and afterwards incubated overnight at 4 °C, with the polyclonal primary antibodies in 1% BSA: rabbit anti-LC3A/B Antibody (1:1000) (Cell-Signaling Technology, USA), mouse anti‐p62 (1:1000) (Abcam, UK), rabbit anti-human caspase 3 (1:700) (Cell Signaling Technology, USA) and mouse anti‐alpha‐actin (Millipore, USA). Secondary antibodies (HRP, anti‐rabbit, anti‐mouse) were from Bio‐Rad, USA (1:5000). Blots were treated with the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, USA) or Clarity Western ECL Substrate (Bio‐Rad, USA). Digital images and densitometry analysis of the bands were obtained in a ChemiDoc XRS System (Bio‐Rad, USA) with Quantity One software V4.6.5 (Bio‐Rad, USA), respectively.