Ab140682

Antibodies to the indicated proteins were purchased from the following vendors: BRD4 (ab128874), BRD2 (ab139690), PSMD14 (ab109123) and PSMD1 (ab140682; all Abcam); BRD3 (A302-368A; Bethyl); tubulin (926-42211; LICOR Biosciences); PSMD4 (3846), PSMD2 (25430), biotin (5597) and H2B (12364; Cell Signaling Technology) and PSMA1 (BML-PW8100-0100; ENZO Life Sciences). IRDye-800 anti-rabbit (926-32211) and IRDye-680RD anti-mouse (926-68072) secondary antibodies were purchased from LICOR Biosciences. Bortezomib (179324-69-7) was purchased from Calbiochem. Streptavidin magnetic beads (88816), NeutrAvidin agarose beads (29200), Lipofectamine RNAiMAX transfection reagent (13778075), RNA-to-C_t 1-Step kit (4392653) and TaqMan gene expression assays were purchased from Thermo Fisher. An RNeasy Plus mini RNA extraction kit (74134) was purchased from Qiagen.

Assays were performed in white ProxiPlate Plus 384-well plates (PerkinElmer) in 25 mM HEPES (pH 7.5), 100 mM NaCl, 100 mM KCl, 0.005% Tween-20, 2 mM MgCl₂, 0.1% BSA and 5 mM ATP. The 26S proteasome (Boston Biochem) was incubated at 2.5 nM with tracer compound MC2_biotin at 5 nM and 0–10 µM assayed compound for 90 min at room temperature. Then, 20 µg ml⁻¹ AlphaScreen streptavidin donor, anti-rabbit acceptor beads (PerkinElmer) and anti-PSMD1 (1 nM; ab140682, Abcam) were added in the semidark, and plates were incubated for an additional 90 min in the dark at room temperature before reading on an Envision plate reader and Envision Manager software (PerkinElmer). Samples were run in at least three technical replicates.