Mouse anti ubiquitin

The following primary antibodies are used: Mouse anti-Actin (Millipore, MAB 1510), Mouse anti-Myc 9E10 (Covance, MMs-150R), Mouse anti-Ubiquitin (Santa Cruz, sc-8017), Mouse anti-GFP (Roche, 11814460001), Rabbit anti-ZDHHC6 (Sigma, SAB1304457), Mouse anti-Transferrin Receptor (Thermo Scientific, 136800), Mouse anti-Flag M2 (Sigma, F3165), Rabbit anti-ANTXR1 (Sigma, SAB2501028), Rabbit anti-TRAPα (Abcam, ab133238), Rabbit anti-Flotillin1 were produced in our laboratory, Mouse anti-CLIMP63 (Enzo, ALX-804–604), Mouse anti-Calnexin (MAB3126), Rabbit anti-GP78 AMFR (Abnova, PAB1684), Rabbit anti-IP3R (Cell signaling, 85685). The following beads were used for immunoprecipitation: Protein G Sepharose 4 Fast flow (GE Healthcare, 17-0618-01), anti-Myc affinity gel (Thermo Scientific, 20169), anti-Flag affinity gel EZview M2 (Sigma, F2426). Drugs were used as follows: Bafilomycin A1 at 100 nM (Sigma, B1793), MG132 at 10 µM (Sigma, C2211), Hydroxylamine at 0.5 M (Sigma, 55459), mPEG-5k at 20 mM (Sigma, 63187), N-ethylmaleimide NEM at 20 mM (Thermo Scientific, 23030), Tris-2- carboxyethyl-phosphine hydrochloride TCEP at 10 mM (Thermo Scientific, 23225), Methyl methanethiosulfonate MMTS at 1.5% (Sigma, 208795), Puromycin at 3 µg/ml (Sigma, P9620).

Antibodies used for immunoblotting, IP, and IHC staining were as follows: Rabbit anti ZHX2 antibody (Genetex, 112232), Rabbit anti HIF1α (Cell Signaling, 36169), Rabbit anti HIF1β (Cell Signaling, 5537), Rabbit anti VHL (Cell Signaling, 68547), rabbit anti HA tag (Cell Signaling, 3724), mouse anti α-Tubulin (Cell Signaling, 3873), mouse anti Ubiquitin (Santa Cruz, sc-8017), and Rabbit anti NF-κB p65 (Cell Signaling, 8242S). Peroxidase conjugated goat anti-mouse secondary antibody (31430) and peroxidase conjugated goat anti-rabbit secondary antibody (31460) were from Thermo Scientific.

Primary antibodies used were: mouse anti-GFAP (Thermo Fisher Scientific), mouse anti-nestin (Rat-401), mouse anti-p62 (Abcam), rat anti-phosphorylated p62 (MBL: D343-3), mouse anti-ubiquitin (Santa Cruz Biotechnology), rabbit anti-TBK1 (Cell Signaling), rabbit anti- phosphorylated TBK1 (Novus Biologicals), rabbit anti-GFAP (Dako), rabbit anti-Ki67 (Spring Bioscience), rabbit anti-p62 (Enzo), rabbit anti-Sox2 (Millipore), rat anti-Ki67 (BioLegend), and guinea pig anti-doublecortin (anti-DCX; EMD Millipore). Secondary antibodies were goat anti–rabbit IgG-FITC, goat anti–rabbit IgG–Texas red, goat anti–mouse IgG-FITC, goat anti–mouse IgG–Texas red, goat anti–mouse IgG-HRP, and goat anti–rabbit IgG-HRP (Jackson Immunology). Dihydroethidium (DHE) was purchased from Sigma-Aldrich and Amlexanox was purchased from MedChemExpress. Transfections were carried out using Lipofectamine 3000 reagent (Invitrogen).

Mashed fly heads were lysed with 10 mM Tris-HCl lysis buffer (pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40 with 1× proteinase inhibitor cocktail [Roche]) for 30 min, and centrifuged at 16,100 g. The supernatant was used for subsequent immunoprecipitation with anti-Myc beads (Chromotek). Beads were washed in TBS buffer with NP-40 (10 mM Tris–Cl (pH7.4), 150 mM NaCl, 0.5 mM EDTA, and 50 nM NP-40 with 1× proteinase inhibitor cocktail) three times, and boiled in SDS loading buffer for standard western blot assays.
For western blot assays, dissected pupa eyes or adult fly heads were homogenized in SDS loading buffer for SDS-PAGE. The blots were probed with primary antibodies against Myc (rabbit, 1:1000; Santa Cruz), mouse anti-β-actin (1:2000; Santa Cruz), mouse anti-ubiquitin (1:1000; Santa Cruz), rabbit anti-GFP (1:2000; Origene), followed by incubation with IRDye 680 goat anti-mouse IgG (1:10000, LI-COR Biosciences) and IRDye 800 goat anti-rabbit IgG (1:10000, LI-COR Biosciences). Signals were detected using an Odyssey infrared imaging system (LI-COR Biosciences).