Nuclear proteasomes carry a constitutive posttranslational modification which derails SDS-PAGE (but not CTAB-PAGE) David S. Pitcher a , Kate de Mattos-Shipley a , Ziming Wang a , Konstantinos Tzortzis a , Katerina Goudevenou a , Helen Flynn b , Georg Bohn a , Amin Rahemtulla a , Irene Roberts a , Ambrosius P. Snijders b , Anastasios Karadimitris a , Maurits F. Kleijnen a, ⁎ a Centre for Haematology, Division of Experimental Medicine, Faculty of Medicine, Imperial College London, Hammersmith Campus, Commonwealth Building 4th Floor, Du Cane Road, London W12 0NN, United Kingdom b London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms, Potters Bar EN6 3LD, United Kingdom abstract article info Article history: Received 29 April 2014 Received in revised form 22 August 2014 Accepted 24 August 2014 Available online 2 September 2014 Keywords: CTAB-PAGE Proteasome ADP-ribose Ubiquitin Nuclear biology Apoptosis We report that subunits of human nuclear proteasomes carry a previously unrecognised, constitutive posttrans- lational modification. Subunits with this modification are not visualised by SDS-PAGE, which is used in almost all denaturing protein gel electrophoresis. In contrast, CTAB-PAGE readily visualises such modified subunits. Thus, under most experimental conditions, with identical samples, SDS-PAGE yielded gel electrophoresis patterns for subunits of nuclear proteasomes which were misleading and strikingly different from those obtained with CTAB-PAGE. Initial analysis indicates a novel modification of a high negative charge with some similarity to polyADP-ribose, possibly explaining compatibility with (positively-charged) CTAB-PAGE but not (negatively- charged) SDS-PAGE and providing a mechanism for how nuclear proteasomes may interact with chromatin, DNA and other nuclear components. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Most denaturing protein gel electrophoresis is done using SDS- PAGE, discontinuous sodium dodecyl sulphate polyacrylamide gel elec- trophoresis [1], due to its ease-of-use and reliability [2]. Although some posttranslational modifications may affect the migration behaviour of proteins on SDS-PAGE, e.g. N-linked glycosylation, SDS-PAGE is believed to, and generally does, faithfully represent the composition of complex protein mixtures [2]. However, we now report a novel modification of nuclear proteasomes that prevents SDS-PAGE from visualising the proteasomal subunits. By way of further background, proteasomes [3,4] are 2.5 MDa protease complexes, located in the cytoplasm and nucleus, which degrade ubiquitin-marked protein substrates. There is an alternative to SDS-PAGE, the much less used CTAB (cetyl trimethyl ammonium bromide)-PAGE system [5,6]. We report that, unlike SDS- PAGE, CTAB-PAGE does visualise these modified subunits of nuclear proteasomes. Thus, for nuclear proteins, combined use of SDS-PAGE and CTAB-PAGE may be required to capture sample complexity. Initial analysis into the nature of the posttranslational modification indicates that the modification shares several characteristics with, but also is dis- tinct from, classical ADP-ribose chains. 2. Materials and methods 2.1. Antibodies, chemicals, enzymes, western blotting, tissue culture The following is a list of commercial antibodies and their corre- sponding manufacturers: α-Rpn10/S5a (MoAb S5a-18, BML-PW9250), α-Rpn12/S14 (PAb, BML-PW8815), α-20S α7 (MoAb LN43, BML- PW8110), α-20S α6 (MoAb MCP20, BML-PW8100), α-Rpt1/S7 (MoAb MSS1-104, BML-PW8825), α-Rpt2/S4 (PAb, BML-PW8305), α-Rpt3/ S6b (PAb, BML-PW8175), α-Rpt4/S10b (MoAb p42-23, PW8830), α-Rpt5/S6a (MoAb TBP1-19, PW8770), α-‘core’ CP subunits (PAb, PW8155), α-β5i/LMP7 (PAb, PW8355), α-ubiquitin (FK2, PW8810), and α-poly(ADP-ribose) (PAb 96-10-04, ALX-210-890-R100) are from Enzo Lifesciences. α-Rpn1/S2 (539166) is from Calbiochem. α-Lamin B2 (MoAb, ab8983) is from Abcam. α-SMARCB1/Ini1 (PAb, sc-13055) is from Santa Cruz. α-Ubiquilin1 (MoAb, 377700) is from Invitrogen. Bortezomib is from Millennium/Takeda. Epoxomicin and Ada- K(Biot)-Ahx3-L3-VS are from Enzo Lifesciences. Doxorubicin, vinblastin, cycloheximide, and epigallocatechin gallate (EGCG) are from Sigma- Aldrich. Micrococcal nuclease, RNaseA/T1, RNaseH and RNAse1 are from Fermentas/Thermo Scientific. Antibodies against yeast proteasome are from Dr. Daniel Finley, Harvard Medical School. The α-Dbf11 anti- body is from Dr. Christian Speck, CSC-MRC London. We generated β- glucanase (Oerskovia xanthineolytica) using an Escherichia coli strain Biochimica et Biophysica Acta 1844 (2014) 2222–2228 ⁎ Corresponding author. E-mail address: m.kleijnen@imperial.ac.uk (M.F. Kleijnen). http://dx.doi.org/10.1016/j.bbapap.2014.08.013 1570-9639/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap