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. Author manuscript; available in PMC: 2019 Jun 21.
Published in final edited form as: Mol Cell. 2018 Jun 21;70(6):1008–1024.e6. doi: 10.1016/j.molcel.2018.06.002

Figure 5. Keap1 negatively regulates UBE2F protein levels.

Figure 5

(A and B) A427 cells were transfected with indicated plasmids, followed by IB (A) and 293 cells were transfected with indicated plasmids and then switched to fresh medium (10% FBS) containing cycloheximide (CHX) post-transfection and incubated for indicated time periods before being harvested for IB (left panels, B) and the band density was quantified (right panel, B).

(C and D) H358 cells were transfected with indicated siRNA oligoes, followed by IB (C) or subjected to half-life study as described above (D).

(E) H358 cells were transfected with pcDNA3 or HA-UBE2M first, and then silenced with siRNA oligoes targeting control, Keap1, and/or CUL3, followed by IB assay by indicated Abs.

(F) H358 cells were transfected with FLAG-Keap1, followed by IP with FLAG Ab and IB with indicated Abs.

(G) H358 cells were transfected with HA-UBE2F-WT or -ETAA mutant, followed by IP with HA- Ab, and IB with Keap1 Ab.

(H–L) The 293 cells were co-transfected with indicated plasmids. HA-His-Ub tagged UBE2F were purified via Ni-NTA affinity, and detected with anti-UBE2F Ab (H, K & L) or followed by IP with HA-Ab and IB with indicated Abs (J); or incubated in a reaction mixture containing ATP, ubiquitin, E1, E2 (UBCH5C), E3 (FLAG-Keap1) and substrate (purified UBE2F protein), followed by ubiquitylation assay and IB with anti-UBE2F Ab (I). WCL: Whole cell extracts.

See also Figure S5.