FIG 6.

DNA damage-induced downregulation of LBR protein levels occurs through the proteasome-mediated pathway and is regulated by RAD6 and PSMD3. (A) The X-ray-induced decrease in LBR depends on proteasome activity, suggesting that the observed LBR downregulation occurs through the proteasome-mediated protein degradation pathway. HEK293T cells treated with 25 μM MG132 for 8 h or not treated were subjected to X-ray irradiation or not irradiated, as indicated, and cells were recovered after another 2.5 h. Cell extracts were prepared and subjected to Western blot assays with the indicated antibodies. (B) X-ray irradiation does not alter the ubiquitination of LBR. HEK293T cells expressing HA-tagged LBR were treated with (+) MG132 for 8 h or not treated (−). Cells were subjected to X-ray irradiation or not irradiated as described above and recovered after another 2.5 h. Cells were harvested and subjected to immunoprecipitation assays under denaturing conditions with anti-HA antibodies. The precipitates were then used for Western blot analyses with the indicated antibodies. (C) The X-ray-induced degradation of LBR depends on the presence of RAD6 and PSMD3. HEK293T cells transfected with a control siRNA, RAD6A/B-specific siRNAs, or a PSMD3-specific siRNA (siPSMD3) were treated with X-ray irradiation or not irradiated, and cells were recovered after 2.5 h. Cell extracts were prepared and subjected to Western blot analyses with the indicated antibodies. (D) PSMD3 interacts with LBR, and their interaction is enhanced by X-ray irradiation. (Top two panels) HEK293T cells were cotransfected with a DsRed2-tagged PSMD3 plasmid and a GFP-tagged LBR plasmid for 48 h. Cell extracts were then prepared and subjected to co-IP assays with anti-GFP antibodies. Western blot analyses were performed with the indicated antibodies. (Bottom) HEK293T cells expressing GFP-LBR and DsRed2-PSMD3 were treated with MG132 for 8 h, and cells were then subjected to X-ray irradiation or not irradiated (control). The cells were recovered after another 2.5 h. Co-IP assays were employed with anti-GFP antibodies, and Western blot analyses were performed with the indicated antibodies. (E) RAD6 is essential for the interaction of PSMD3 and LBR under X-ray irradiation conditions. HEK293T cells expressing GFP-LBR and DsRed2-PSMD3 were transfected with a control siRNA (−) or RAD6A/B-specific siRNAs (+) for 48 h. Cells were then treated with MG132 for 8 h and were subjected to X-ray irradiation (+) or not irradiated (−). Cells were recovered after another 2.5 h. Cell extracts were prepared and subjected to co-IP assays with anti-GFP antibodies, followed by Western blot analyses with the indicated antibodies. IgGH, IgG heavy chain.