Figure 4. K63-linked ubiquitination of BCL10 is mediated by RNF8–UBC13 complex. (A) BCL10 is modified with K63-linked ubiquitins. RNF8-depleted or mock-depleted 293T cells were co-transfected with FLAG-Vec or FLAG-BCL10 along with HA-ubiquitin, HA-ubiquitin K48 only (HA-Ub[K48[), or HA-ubiquitin K63 only (HA-Ub[K63]). Total cell lysates extracted 48 h after transfection were subjected to immunoprecipitation with an anti-FLAG antibody and immunoblotting with antibodies as indicated. (B) BCL10(T91A) was not ubiquitinated in vivo. 293T cells were transiently co-transfected with HA-ubiquitin and FLAG-Vec, FLAG-BCL10, or FLAG-BCL10(T91A). Immunoprecipitates with an anti-FLAG antibody were probed with antibodies as indicated. (C) Depletion of RNF8 reduced etoposide-induced focus formation of BCL10. HeLa cells were transfected twice with either RNF8 siRNAs or a non-targeting control siRNA (si-CTR). Forty-eight hours after the second transfection, cells were treated with etoposide for 1 h before they were fixed with 4% paraformaldehyde. Immunofluorescence staining with anti-BCL10 and anti-γ-H2AX was performed as described in the Methods. The efficiency of RNF8 knockdown was determined by immunoblotting with antibodies as indicated. Scale bars: 20 μM. (D) Depletion of UBC13 reduced etoposide-induced focus formation of BCL10. Experiments were performed as described in (C) except that UBC13 was depleted in HeLa cells.