Rabbit anti atg3

Total protein (30 μg) and pre-stained molecular weight markers were separated by SDS-PAGE and electro-transferred onto a polypropylene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked by incubating with Tris-buffered saline containing 0.1% Tween-20 and 5% non-fat milk for 2 h at room temperature. Then, the membranes were probed at 4°C overnight with the following primary antibodies: from Cell signaling (Danvers, MA, USA): rabbit anti-Atg3 (catalog no.:3415; 1:1000 dilution), rabbit anti-LC3B (12741; 1:1000), rabbit anti-GAPDH (5174; 1:1000), rabbit anti-p62 (8025; 1:1000), rabbit anti-p-AKT (S473) (9271; 1:1000), rabbit anti-p-mTOR (S2448) (5536; 1:1000), rabbit anti-caspase-3 (9662; 1:1000) and mouse anti-caspase-8 (9746; 1:1000) antibodies; from Zen BioScience (Chengdu, Sichuan, China): mouse anti-FLAG (390002; 1:2000). After washing, the membrane was incubated with a secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. Finally, the membrane was prepared with enhanced chemiluminescence substrate (Pierce, Rockford, IL, USA) and visualized on a LAS-3000 Luminescence Image Analyzer (Fujifilm, Tokyo, Japan). All antibodies were purchased from Cell Signaling Tech Inc.

Monoclonal Abs specific for CD1a, CD14, CD86 (BD Bioscience, San Jose, CA, USA) and Fixable Viability Dye (FvDye, eBioscience, San Diego, CA, USA) were used as direct conjugates to fluorescein isothiocyanate (FITC), phycoerythrin (PE) or eFluor780 as needed.
For immunoblotting analysis, goat anti-Atg7 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), rabbit anti-Atg3 (Cell Signaling, Danvers, MA, USA), rabbit anti-GABARAP (Santa Cruz Biotechnologies), rabbit anti-LC3 (Cell Signaling), mouse anti-actin (Sigma-Aldrich, St. Louis, MO, USA), and horseradish peroxidase-conjugate secondary anti-goat, anti-mouse and anti-rabbit Abs (Santa Cruz Biotechnology) were used.
Where indicated, LPS (Sigma-Aldrich) was used at 1 μg/ml.