Figure 3.

BLM is ubiquitylated by RNF8 and RNF168 in vivo. (A) Loss of RNF8 leads to decreased K63-linked ubiquitylation of BLM in vivo. U2OS shRNF8 cells were grown in the absence or presence of Doxycycline (Dox), without or with HU co-treatment. (Top) Nuclear extracts were probed with antibodies against RNF8, BLM (A300-110A) and Lamin A/C. (Middle and bottom) Immunoprecipitations were carried out with anti-Ubiquitin K63-linkage specific antibody (or the corresponding IgG). The immunoprecipitates were probed with antibodies against BLM (A300-120A). (B, C). Loss of RNF168 leads to decreased K63-linked ubiquitylation of BLM in vivo after HU treatment. Same as (A) except U2OS shRNF168 cells were used in (B) and RIDDLE syndrome cells complemented with either empty vector or HA-tagged RNF168 cells were used in (C). U2OS shRNF168 cells were grown in the absence or presence of Doxycycline (Dox). Both U2OS shRNF168 and RIDDLE syndrome cells were treated with HU. The direct westerns are on top while immunoprecipitation with anti-Ub (K63) antibodies (or the corresponding IgG) followed by anti-BLM (A300-120A) westerns is shown at the bottom.

Source data for this figure is available on the online supplementary information page.

Source data for Figure 3 ^{(2.2MB, pdf)}