Figure 6. DJ-1/Parkin is the major E3 for UBE2F ubiquitylation.
(A) H1299 cells were transfected with indicated plasmids, followed by IP and IB with indicated Abs. WCL: Whole cell extracts.
(B) H1299 cells were treated with indicated compounds for 24 h, followed by IP with UBE2M Ab and IB with indicated Abs. WCL: Whole cell extracts.
(C and D) A427 cells were transfected with indicated siRNAs oligoes, followed by IB with indicated Abs (C), or subjected to half-life study with cycloheximide (CHX) (D). The band density was quantified using ImageJ software and plotted (bottom panel, D).
(E and F) A427 cells were transfected with indicated plasmids, followed by IB with indicated Abs, or subjected to protein half-life study with CHX (F) and the band density was quantified.
(G) H358 cells were transfected with siRNA oligoes targeting control, DJ-1 and/or Parkin, and then treated with DMSO or CoCl2 for 24 h, followed by IB assay.
(H&I) H1299 cells were co-transfected with indicated plasmids. Cells were treated with DMSO vehicle control, or three indicated compounds (I). HA-His-Ub tagged UBE2F were purified via Ni-NTA affinity, and detected with anti-UBE2F Ab
(J) Pure E1, E2 (UBE2M or UBCH5C), E3 (Parkin and/or Pink1), and substrate (UBE2F) were incubated in a reaction mixture containing ATP and ubiquitin for 1 h, followed by IB with UBE2F Ab.
See also Figure S6.