Figure 2.

HR23B has an impact on the ubiquitin proteasome system. (a) (i) U2OS cells stably expressing inducible ectopic Flag-HR23B were transfected with His₆-ubiquitin (His-Ub; 2 μg), then treated with doxycycline (+ and −, 1 μg/ml) for 24 h following an additional 20 h treatment with SAHA (5 μM) and/or 4 h treatment with MG132 (20 μM) as indicated. Cell lysates and Ni²⁺ pull-down eluates were analysed by immunoblotting with anti-His as described. The input HR23B is shown underneath, together with actin levels. (ii) Quantitation of the ubiquitin signal. (b) (i) U2OS cells were transfected with control (NT) or HR23B siRNA (50 nM) for 24 h, then transiently transfected with His₆-ubiquitin (His-Ub; 2 μg) for 24 h, following an additional 20 h treatment with SAHA (5 μM) and/or 4 h treatment with MG132 (20 μM) as indicated. Ni²⁺ pull-down eluates were analysed by immunoblotting with anti-His. The input HR23B level is shown underneath, together with actin levels. (ii) Quantitation of the ubiquitin signal. (c) (i) U2OS cells expressing inducible ectopic Flag-HR23B were transiently transfected with GFP^u or GFP (100 ng), then treated with or without doxycycline (+ or −, 1 μg/ml) for 24 h as indicated. Cell lysates were analysed as described. Levels of GFP, HR23B and actin are shown. (ii) Quantitation of the GFP signal. (d) (i) U2OS cells were transfected with control (NT) or HR23B siRNA (50 nM) for 24 h, then transiently transfected with GFP or GFP^u (100 ng) for 48 h as indicated. Cell lysates were analysed as described above. Levels of GFP, HR23B and actin are shown. (ii) Quantitation of the GFP signal