Fig. 6: The presence of hRpn13Pru is cell type dependent.
a Lysates from RPMI 8226 WT, HCT116 WT, or HCT116 trRpn13 cells were immunoprobed for hRpn13 with 1 s or 5 min exposure times and β-actin as indicated. b LC-MS analysis of GST-hRpn2 (940–953) (control, left panel) or GST-hRpn2 (940–953)-pulldown sample from lysates of RPMI 8226 WT cells (right panel). The mass spectra (upper panel) were deconvoluted from the UV peak (lower panel) indicated with a black arrow. c Ribbon representation of the structure of hRpn13 Pru domain (solid black line, PDB 5IRS) and full-length hRpn13 (dashed gray line, PDB 2KR0) to highlight the greater accessibility to XL5 following loss of the Pru-interacting DEUBAD domain. d Lysates from Hs27, SK-OV-3, MM.1S, NCI-H929, or RPMI 8226 WT cells were immunoprobed for hRpn13 and β-actin as indicated. e MM1.S cells were treated with 2.5 or 5 μM of XL5-VHL-2 for 48 h and cell metabolism measured by an MTT assay; data represent mean ± SD of n = 6 biological replicates. Viability is calculated as (λ570)sample/(λ570)control*100 (%). f Lysates from RPMI 8226 WT and trRpn13-MM2 cells transfected for 48 h with empty vector (EV) or plasmids expressing FLAG-hRpn13 full length or FLAG-hRpn131–279 proteins were treated for 24 h with 40 μM XL5-VHL-2 or DMSO (vehicle control) and immunoprobed as indicated with antibodies against hRpn13, cleaved caspase-9, and β-actin. Immunoprobing for cleaved caspase-9 and hRpn13 was done separately with re-probing for β-actin. g Volcano plot displaying proteomic changes caused by XL5-VHL-2 treatment determined by quantitative TMT proteomics analysis performed on lysates from RPMI 8226 trRpn13-MM2 cells treated for 24 h with DMSO (control) or 40 μM XL5-VHL-2 in triplicate. p value was calculated by two-tailed two-sample equal variance t test. A dashed line indicates the value −log100.05.
