Fig. 5: Chemical probing by XL5-PROTACs reveals a DEUBAD-lacking hRpn13 species.

a Immunoblot of whole-cell extract from RPMI 8226 WT cells treated for 24 h with 40 μM XL5, 40 μM XL5-PROTAC or DMSO (vehicle control) detecting hRpn13 (1 s or 3 min exposure) or β-actin. b Immunoblots with antibodies against hRpn13 or GST as indicated from GST pulldowns of RPMI 8226 WT cell lysates. Pulldowns were done with GST-hRpn2 (940–953) or GST (control). c RPMI 8226 WT cells were immunoprecipitated with anti-hRpt3 or IgG (control) antibodies and immunoprobed for hRpn13, hRpn2, and hRpt3 as indicated. d Lysates from RPMI 8226 WT cells treated with indicated concentration of XL5-VHL-2 or DMSO (control) for 24 h were immunoprobed for cleaved caspase-9, hRpn13 (1 s or 40 s exposure), or β-actin (as a loading control). A black asterisk indicates cleaved caspase-9 in the 40 s immunoblot for hRpn13, as hRpn13 was probed following cleaved caspase-9 and without stripping the membrane (top panel). e Immunoblots of whole-cell extract from RPMI 8226 WT cells treated for the indicated hours with 40 μM XL5-VHL-2 or DMSO (0 h, vehicle control) detecting hRpn13 or β-actin. Percentage (%) is calculated as the ratio of intensities for hRpn13^Pru normalized to β-actin (\({I}_{{hRpn {\it{{13}}}}^{Pru}}\)/I_β-actin)_sample divided by that of DMSO-treated cells and multiplied by 100. Percentage (%) derived from left (d) or top (e) panel immunoblots were plotted against XL5-VHL-2 concentration (d, μM) or time (e, hours) and fit by using the equation [Inhibitor] vs. normalized response - Variable slope (d) and One phase decay (e) in GraphPad Prism8. Half-degrading concentration (DC₅₀, d), maximal degradation (D_max, d), and half-life (t_1/2, e) values are included.