Ubiquitin bml pw8810

The following antibodies were used for western blot: HAX1 (BD Transduction Laboratories: 610825, 1:500), cIAP2 (ABCAM: ab32059, 1:1000), cIAP1 (R&D Systems: AF8171, 1:500), FLAG (F1804, 1:5000), His (H1029, 1:3000), and α-tubulin (T6074, 1:5000) (Sigma-Aldrich Co. LLC.), p100/p52 (#3017, 1:1000), NIK (#4994, 1:1000), and cIAP2 (#3130, 1:1000) (Cells Signaling Technology, Inc.), GFP (sc-9996, 1:1000) and HA (sc-805, 1:1000) (Santa Cruz Biotechnology, Inc.), and ubiquitin (BML-PW8810, 1:100, Enzo Life Science). Recombinant human CD40 ligand/TNFSF5 (617-CL) and recombinant human LIGHT/TNFSF14 (664-LI) were obtained from R&D Systems. Pierce anti-HA agarose (26181) was obtained from Thermo Scientific. Anti-FLAG M2 affinity gels (A2220), 3× FLAG peptide (F4799), anti-c-Myc agarose affinity gels (A7470), cycloheximide (C4859), and MG132 (C2211) were purchased from Sigma-Aldrich Co. LLC. Bortezomib (velcade) (MG-341) and protein G plus/protein A agarose suspension (IP05) were purchased from Merck Millipore. Complete protease inhibitor cocktail (13760700) was purchased from Roche.

Equal concentrations (12–25 μg) of isolated RBC membranes (n = 7 per group) or vesicles (n = 5 per group) were loaded in Laemmli gels and transferred to nitrocellulose membranes. Primary monoclonal and polyclonal antibodies for a variety of proteins were used, along with species-specific secondary antibodies conjugated with HRP. Antibodies to the following proteins were used: band 3 (B 9277) from Sigma-Aldrich (Munich, Germany); ubiquitin (BML-PW8810) from Enzo Life Sciences (New York, NY, USA); HSP70 (sc-1060R) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and caspase-3 (#9662) and DJ-1 (#5933) from Cell Signaling Technology (Danvers, MA, USA). Antibodies against stomatin and 4.1R were kindly provided by Prof. R. Prohaska (Institute of Medical Biochemistry, University of Vienna, Austria) and Prof. J. Delaunay (Laboratoire d’ Hématologie, d’ Immunologie et de Cytogénétique, Hopital de Bicetre, Le Kremlin-Bicetre, France), respectively. The immunoblots were developed through chemiluminescence, and the bands were quantified by scanning densitometry (Gel Analyzer v.1.0, Athens, Greece). To estimate oxidative modifications of membrane and vesicular proteins, the Oxyblot kit was used, per the manufacturer’s specifications (Oxyblot, Millipore, Chemicon, Temecula, CA, USA). The proteome carbonylation index (PCI) was then calculated [68 (link)].

Antibodies recognising FAM83B (SA270), FAM83D (SA102), FAM83F (SA103), FAM83H (SA273), CK1α (SA527), CK1ε (SA610), CK1δ (SA609), and GFP (S268B) were generated in-house and are available for request from the MRC-PPU reagents website (http://mrcppureagents.dundee.ac.uk). Antibodies recognising GAPDH (14C10) (#2118), IKZF1 (D6N9Y) (#14859), CRBN (D8H3S) (#71810), Na, K-ATPase alpha1 (D4Y7E) (#23565), and Lamin A/C (#2032) were obtained from Cell Signalling Technology. Additional antibodies used were FAM83G (ab121750; Abcam), α-tubulin (MA1-80189; Thermo Fisher Scientific), Ubiquitin (BML-PW8810; Enzo), HIF-1α (6109590; BD Biosciences), and ZFP91 (A303-245A; Bethyl Laboratories). Secondary antibodies used were StarBright Blue 700 goat anti-rabbit IgG (12004161; Bio-Rad), StarBright Blue 700 goat anti-mouse IgG (12004158; Bio-Rad), IRDye 800CW donkey anti-goat IgG (926-32214; LI-COR) and IRDye 800CW goat anti-rat IgG (926-32219; LI-COR).