Fig. 3

SUMO pathway is required for Daxx recruitment to centromeres. a Both SUMO-1 and SUMO-2 are accumulated with Daxx at centromeres. SUMO-1 (green, upper row) and SUMO-2 (green, bottom row) co-localize with Daxx (red) at centromeres (CREST, blue) in HEp2 cells after treatment with proteasome inhibitor MG132. b Expression of SUMO de-conjugation enzymes SENPs affects Daxx accumulation at centromeres. Representative images of HEp2 cells transiently expressing GFP (left), GFP-SENP1 (middle) or GFP-SENP2 (right) and treated with MG132, 4 h. SENP1-depleted Daxx (red) from centromeres (CREST, blue) and dispersed Daxx homogenously in nucleoplasm; SENP2-depleted Daxx from centromeres and co-localized with Daxx in ND10-like domains. c Daxx interaction with CENP-B is SUMO-2 dependent. FLAG-HA-CENP-B expressing HEp2 cells were transfected with scrambled siRNA (CTL), SUMO-1 or SUMO-2 siRNA (Additional file 4: Fig. S4, top for the levels of SUMO-1 and SUMO-2 depletion) and treated with MG132. Chromatin fraction of cell lysates (Additional file 4: Fig. S4, bottom for Daxx input) was IP with anti-FLAG-M2 magnetic beads. Eluates were immunoblotted with HA (for CENP-B) and Daxx. Relative to control siRNA, SUMO-2 depletion reduced Daxx co-IP with CENP-B to 0.2, while depletion of SUMO-1 had minor effect reducing Daxx pull-down to 0.7