Figure 6.
RAP80 regulates BLM stability. (A, B) BLM and RAP80 interact in vivo. Immunoprecipitation of BLM was carried out in (A) BS/A-15 cells or only in (B) A-15 cells using either (A) anti-RAP80 or (B) anti-BLM (NB 100–161) antibody or their corresponding IgG. Cells were grown in three conditions, namely Asynchronous (Asyn.), after HU treatment (+HU) and in postwash (+HU/PW) condition. Western blotting on the immunoprecipitates was carried out with antibodies against anti-BLM (NB 100-161) and anti-RAP80. (C) BLM and RAP80 colocalize after replication arrest. GFP-BLM cells were either grown under asynchronous conditions or treated with HU (+HU). GFP-BLM cells were co-stained with anti-RAP80 antibody. Scale 5 μM. (D) RAP80 interacts with BLM via its UIM after exposure to HU. 293T cells were transfected with either EGFP-tagged wild-type RAP80 or its ΔUIM mutant. (Left) Whole cell lysates were probed with antibodies against BLM (A300-110A) or GFP. CR band represents a cross-reactive band. (Right) Immunoprecipitations were carried out with antibodies against RAP80 or IgG control. Immunoprecipitates were probed with antibodies against BLM (A300-110A) and RAP80. IgG acted as a control for the usage of equal amount of antibody during immunoprecipitation. (E) Overexpression of RAP80 stabilizes BLM. 293T cells were either left untransfected or transfected with either Flag-BLM or GFP-RAP80 or both. Whole cell lysates were probed with antibodies against BLM (NB 100-161), GFP and hsp90. (*) is a cross contaminating band. (F) Ablation of RAP80 destabilizes BLM. GFP-BLM cells were transfected with siRNA control, siRNA BLM, siRNA RAP80 or siRNA BLM and siRNA RAP80. Whole cell lysates were probed with antibodies against BLM (A300-110A), RAP80 and hsp90. (G, H) Destabilization of BLM level after ablation of RAP80 is reversed after treatment with proteasome inhibitors. GFP-BLM cells were either transfected with either siRNA control, or siRNA RAP80 and grown in the absence or presence of (G) LLnL or (H) MG132. Whole cell lysates were probed with antibodies against BLM (A300-110A), RAP80 and hsp90. (I) BLM physically interacts with RAP80. (Top) Glutathione-Sepharose bound GST or GST-BLM (1–1417) was incubated with equal amounts of the lysates from 293T cells transfected with GFP-RAP80. The bound GFP-RAP80 was probed with antibody against GFP. (Bottom) Coomassie gel indicating purified GST and GST-BLM (1–1417). (J) Interaction between GST-BLM and the internal deletions and end fusion proteins of RAP80. (Left) Western blot depicting the expression of the wild-type RAP80 and the RAP80 internal deletions and the end fusion constructs as detected after probing with antibodies against GFP. (Right) Bead bound purified GST-BLM was incubated with lysates expressing EGFP-tagged RAP80 mutants. Bound RAP80 was detected by probing with anti-GFP antibody. (*) represents the EGFP-RAP80 fragments, which interact with GST-BLM.
Source data for this figure is available on the online supplementary information page.
