Rabbit polyclonal anti lc3b antibody

Bicinchoninic acid (BCA) protein assay kit (P0009), LDH cytotoxicity assay kit (C0016), enhanced chemiluminescence (ECL) kit (P0018M), MTT cell proliferation and cytotoxicity assay kit (C0009), Hoechst staining kit (C0003), benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD) (C1202), were obtained from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Glutathione (GSH) assay kit was obtained from Nanjing Jiancheng Bioengineering Institute (Jiancheng, Nanjing, Jiangsu, China). Hydrogen peroxide (H₂O₂) and 3-methyladenine (3-MA) were obtained from Sigma-Aldrich (St. Louis, USA). Rabbit monoclonal anti-caspase-3 (cleaved) antibody was obtained from Beyotime Institute of Biotechnology. Rabbit polyclonal anti-LC3B antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit or -mouse secondary antibodies were purchased from Sigma-Aldrich. Mouse monoclonal anti-β-actin antibody and rabbit polyclonal anti-ATG5 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). X-tremeGENE siRNA transfection regent was from Roche (Basel, Switzerland).

NSC34 cells were plated in 12-well multiwell plates containing coverslips at 70 000 cells/well density, transiently transfected with the plasmid coding for GFP-ARQ48 as previously described and treated with 100 mm trehalose and/or 100 nm Bicalutamide and/or 10 nm testosterone for 48 h. The cells were then fixed and processed as previously described (Sau, 2007). To analyze LC3 and SQSTM1/p62 protein expressions, we used the following primary antibodies: rabbit polyclonal anti-LC3-B antibody 1 : 1000 (Sigma-Aldrich) in 5% nonfat milk, rabbit polyclonal anti-SQSTM1/p62 antibody 1 : 1000 (Abcam, Cambridge, UK) in 5% nonfat milk. Secondary antibodies used were: Alexa 488 anti-rabbit and Alexa 594 anti-rabbit (Life Technologies) 1 : 1000 in milk. Cells were stained with DAPI to visualize the nuclei. An Axiovert 200 microscope (Zeiss Instr., Oberkochen, Germany) equipped with FITC/TRITC/DAPI and combined with a Photometric Cool-Snap CCD camera (Ropper Scientific, Trenton, NJ, USA) was used. Images were processed using the Metamorph software (Universal Imaging, Downingtown, PA, USA).

Thapsigargin (Tg), mouse monoclonal anti-Flag M2 antibody, rabbit polyclonal anti-LC3B antibody, rabbit polyclonal anti-p62/SQSTM1 antibody, and mouse monoclonal anti-actin antibody were purchased from Sigma-Aldrich. Mouse monoclonal antibodies against GRP78, GRP94, ATF6, ATF4, Beclin-1, PERK, phospho-PERK, eIF2α, and phospho-eIF2α were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal IRE 1 and phospho-IRE 1 were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit or -mouse secondary antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Alexa Fluor 594-labeled donkey anti-goat IgG was purchased from Life Technologies (Carlsbad, CA, USA). 4′,6′-diamidino-2-phenylindole (DAPI) and Cy3-labeled goat anti-mouse IgG were purchased from Thermo Scientific (Waltham, MA, USA).