Figure 4

hHR23a rescues and stabilizes BCA2 from UbcH5 mediated poly-ubiquitination. [A] Immunoblots of lysates from HEK293T cells cotransfected with GST-tagged BCA2 (GST-BCA2) and BCA2 with the RING domain mutation C228/231A (GST-BCA2-RING mt) and/or expression vectors for His-tagged Ubc3 and UbcH5a/b/c as indicated. [B] Western blots of HEK293T cells which were co-transfected with stable amounts of either GST-tagged wild-type BCA2 or GST-BCA2 RING mt along with an increasing amount of myc/his-hHR23a. Relative intensity of BCA2 normalized to β-Tubulin is given below the blot as fold change compared to lanes 1 and 5 respectively. [C] Immunoblot showing lysates of in vivo ubiquitination assay probed with anit-HA following immunoprecipitation of Flag-BCA2 with anti-FLAG antibodies. HEK293T cells were co-transfected with HA-ubiquitin, and FLAG-BCA2 along with either myc/his-UbcH5b or myc/his-hHR23a or both as indicated above lanes. [D] Immunoblot of a half-life assay of HEK293T cells transfected with GST-BCA2 with and without myc-hHR23a. Cells were treated over a 24 hours time course with cyclohexamide. Relative intensity of BCA2 normalized to β-Tubulin is given below the blot as fold change.