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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 4. CUL3 negatively regulates UBE2F protein levels.

Figure 4

(A and B) H358 cells were transfected with siRNAs oligoes targeting different cullins (A) or either indicated plasmids (B), followed by IB with indicated Abs.

(C and D) A427 cells were transfected with siRNA oligo targeting CUL3 (C) or plasmid expressing CUL3 (D), then switched to fresh medium (10% FBS) containing cycloheximide (CHX) post-transfection and incubated for indicated time periods before being harvested for IB (upper panels, C and D) and the band density was quantified (bottom panels, C and D).

(E and F) H358 cells were transfected with indicated plasmids and cells lysates were then immunoprecipitated using HA antibody (E) or Myc antibody (F), followed by IB with indicated Abs. WCL: Whole cell extracts.

(G–J) 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 (G & I), or incubated in a reaction mixture containing ATP, ubiquitin, E1, E2 (UBCH5C), E3 (Myc-tagged CUL3) (H) or various Myc-tagged cullins (J) and substrate (purified UBE2F protein), followed by ubiquitylation assay and IB with anti-UBE2F Ab.

See also Figure S4.