Anti gabarap atg8a

The primary antibodies used were the anti‐Ubiquitin (Santa Cruz Biotechnology, Inc., sc‐8017), anti‐foxo (COSMO BIO CO, CAC‐THU‐A‐DFOXO), anti‐26S proteasome α (Santa Cruz Biotechnology, Inc., sc‐65,755), anti‐26S Proteasome p54 (Rpn10) (Santa Cruz Biotechnology, Inc., sc‐65,746), anti‐pAMPK (Cell Signaling Technology, #2535), anti‐GABARAP (Atg8a) (Cell Signaling Technology, #13733), anti‐Lamp1 (Abcam, ab30687), anti‐ATP5A (Abcam, ab14748), anti‐Actin (Cell Signaling Technology, #8457), and anti‐Gapdh (Sigma‐Aldrich, G9545). The anti‐ref(2)P/p62 antibody was kindly donated from Prof. G. Juhász. The secondary antibodies Peroxidase AffiniPure Donkey anti‐Mouse IgG (715‐035‐150) and Peroxidase AffiniPure Donkey anti‐Rabbit IgG (711‐035‐152) were purchased from Jackson ImmunoResearch Laboratories, Inc. The anti‐Rabbit‐IgG Alexa Fluor 647 (711‐605‐152), anti‐Rabbit‐IgG Alexa Fluor 488 (111‐545‐003), anti‐Mouse‐IgG Rhodamine (TRITC) AffiniPure (715‐025‐151), anti‐Mouse‐IgG Alexa Fluor 488 (115‐545‐003), and anti‐Mouse IgG DyLight™ 405 (715‐475‐151) antibodies were also from Jackson ImmunoResearch Laboratories, Inc. Ponceau S solution (6226–79) was from Sigma‐Aldrich.

Protein extracts were prepared in Laemmli loading buffer containing 2.5% beta-mercaptoethanol (except for experiment in non-reducing condition where no beta-mercaptoethanol was added) and heated for 5 min at 95°C before separation of 20 μg total proteins on 8 or 12% SDS-PAGE gels. Separated proteins were transferred onto nitrocellulose or PVDF membranes. The membranes were blocked in TBS (Tris-buffered saline; 50 mM Tris-Cl, pH 7.6, 150 mM NaCl), 0.1% Tween-20, 5% non-fat milk. The following antibodies were used: anti-GFP (Santa Cruz sc-9996, 1:1,000), anti-GABARAP/Atg8a (Cell Signaling Technology No. 13733, 1:2,000), anti-Ref(2)P (Abcam ab178440, 1:1,000), anti-β actin (Abcam ab8227, 1:2,000), anti-α tubulin (Sigma-Aldrich T5168, 1:40,000), HRP-coupled secondary antibodies anti-rabbit and anti-mouse (Thermo Scientific No. 31460 and 31450, 1:10,000). Signals were developed using the ECL detection reagents (Amersham, RPN2209).