Fig. 4: UBL7 binds to VCP complex through direct interaction with UBE4B.
a GO enrichment analysis of UBL7 binding proteins identified by FLAG-IP and mass spectrum in Ubl7flag/flag testis and spermatids. BP, biological processes. Source data are provided as a Source Data file. b WB validation of proteins identified by FLAG-IP in Ubl7flag/flag testis. Endogenous protein co-precipitated with FLAG-UBL7 were detected by indicated antibodies. c Schematic of functional domains and truncated forms of UBL7 protein. d IP of truncated forms of UBL7 and UBE4B. Flag-tagged whole-length or truncated forms of UBL7 were co-transfected with Myc-tagged UBE4B in 293 T cells. 48 h after transfection, cell lysates were subjected to IP with FLAG antibody. UBE4B co-precipitated with FLAG-UBL7 was detected by anti-Myc antibody. Cells transfected with FLAG-tag vector serve as a negative control. e IP of UBE4B and VCP. FLAG-UBE4B was co-transfected with HA-VCP or empty HA-tag vector in 293 T cells and subjected to IP with anti-HA antibody. UBE4B co-precipitated with HA-VCP was detected by FLAG antibody. f IP of UBL7 and VCP in presence of UBE4B in 293 T cells. FLAG-UBL7 was co-transfected with HA-VCP in presence or absence of Myc-UBE4B in 293 T cells. UBE4B and VCP co-precipitated with UBL7 was detected by Myc and HA antibody, respectively. g IP of UBL7 and its truncated forms with Ub-conjugates. FLAG-tagged full-length or truncated UBL7 were transfected in 293 T cells. 48 h after transfection, cells transfected with full length UBL7 were treated with MG132 (10 μM) or TAK243 (10 μM) for 1 h. Then cells were lysed for FLAG-IP. Ub-conjugates co-precipitated with FLAG-UBL7 was detected with anti-ubiquitin antibody. FLAG-GFP serves as a negative control. h A model shows that UBL7 interacts with UBE4B and indirectly binds to VCP complex. In the meantime, UBL7 binds to Ub-conjugates extracted and processed by the VCP complex.
