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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 3. UBE2M negatively regulates UBE2F protein levels.

Figure 3

(A) A427 cells were firstly transfected with si-Cont or si-UBE2M-2 and then treated with indicated compounds. Cells were harvested for IB with indicated Abs.

(B and C) H358 cells were transfected with HA-tagged UBE2M for 48 h, followed by IB with indicated Abs (B), or switched to fresh medium (10% FBS) containing cycloheximide (CHX) and incubated for indicated time periods before being harvested for IB (top panels, C), and the band density was quantified using ImageJ software and plotted (bottom panel, C).

(D and E) H358 cells were transfected with two independent siRNAs targeting UBE2M or si-Cont for 48 h, followed by IB with indicated Abs (D), or were transfected with si-UBE2M-2 and then switched to fresh medium (10% FBS) containing cycloheximide (CHX) post-transfection and incubated for indicated time periods before being harvested for IB (top panels, E) and the band density was quantified (bottom panel, E).

(F) A427 and H358 cells were transfected with two independent siRNAs targeting UBE2F or si-Cont for 48 h, followed by IB with indicated Abs.

(G) UBE2F and UBE2M bind to each other physically. H358 cell lysates were immunoprecipitated using UBE2M Ab (left), or UBE2F Ab (right), followed by IB to detect endogenous proteins. The 10% of the extracts was loaded as the input.

(H and I) The 293 cells were co-transfected with indicated plasmids or treated with or without MG132. Ni-NTA affinity-purified fractions (top panel) were analyzed by IB with anti-UBE2F (H) or anti-UBE2M (I); 10% of Ni-NTA affinity-purified fractions or whole cell extracts (bottom panels) were analyzed by HA antibody to detect the exogenous Ub, UBE2F, or UBE2M.

(J) H1299 cells were cotransfected with HA-His-Ub and HA-UBE2F, and then transfected with si-Cont or si-UBE2M-2, followed by treatment with indicated compounds for 24 h. Cell lysates were subjected to Ni-NTA purification, followed by IB with anti-UBE2F Ab (top panel). Whole cell lysates were subjected to IB with indicated Ab (bottom panels).

See also Figure S3.