Anti-ATG5 (C-terminal) antibody produced in rabbit

Protocol tips

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	Dilute primary Ab at 1:100 and incubate overnight

Protocol tips
Dilute primary Ab at 1:100 and incubate overnight

Publication protocol

Rat spinal cord tissue (5 mm) centered on the injury site were lysed and homogenized in RIPA buffer (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) supplemented 1x protease inhibitor cocktail (Sigma-Aldrich Co. LLC), phosphatase inhibitor cocktail II and III (Sigma-Aldrich Co. LLC), sonicated and centrifuged at 20 000 g for 20 min. Protein concentrations were determined by the Pierce BCA method (ThermoFisher Scientific, Waltham, MA, USA). Samples were run on 4–15% or 15% SDS-PAGE (Bio-Rad, Hercules, CA, USA), and transferred to PVDF membrane (Bio-Rad). Membranes were incubated with primary antibodies overnight, with HRP-conjugated secondary antibodies for 1 h, visualized using SuperSignal West Dura Extended Duration Substrate (ThermoFisher Scientific), and imaged with ChemIDoc TM MP system (Bio-Rad). The signal was quantified by Image Lab software (Bio-Rad). Primary antibodies: LC3 (1 : 200, Novus, Cat. No. NB100-2220, Littleton, CO, USA), p62 (1 : 200; Progen, Cat. No. GP62-C, Heidelberg, Germany), Beclin1 (1:1000; Santa Cruz Biotechnology, Cat. No. sc-11427, Dallas, TX, USA), phospho-ULK1 (1:1000; Cell Signaling, Cat. No. 5869, Danvers, MA, USA), VPS34 (1 : 1000, Sigma-Aldrich, V9764), ATG5 (1 : 1000; Sigma-Aldrich, Cat. No. A0731), β-actin (1:10,000; Sigma-Aldrich, Cat. No. A1978), Ubiquitin (1 : 1000; Cell Signaling, Cat. No. 3936), CTSD (1 : 100, Santa Cruz Biotechnology, Cat. No. sc-6486), Spectrin (1 : 5000; Enzo Life Science International, Cat. No. BML-FG6090, Farmingdale, NY, USA), cleaved caspase 3 (1 : 500, Cell Signaling, Cat. No. 9661), caspase 12 (1 : 200, Cell Signaling, Cat. No. 2202), GRP78 (1 : 500, Cell Signaling, Cat. No. 3177), CHOP (1 : 1000, Cell Signaling, Cat. No. 2895), ATF4 (1 : 1000, Sigma Aldrich, Cat. No. WH0000468M1), and LAMP2 (1 : 1000, Cat No. ABL-93) (developed by J. Thomas August and obtained from Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242, USA).

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Manufacturer protocol

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