Figure 5.

RNF128 Iso1 binds tighter with UBCH5 family members. (A) V5-tagged RNF128 (either Iso1 or Iso2) were co-transfected either with UBCH5A or UBCH5C as indicated in CpA cells. Twenty hours after transfection, cells were treated with proteasomal inhibitor MG132 (2 μmol/L for 4 hours) before harvest and immunoprecipitation followed by immunoblotting using indicated antibodies. (B) To test the interaction ability between RNF128 Iso1 and Iso2 with either WT or catalytic-dead (C85A) mutant UBCH5A, we performed co-transfection, immunoprecipitation, and immunoblot analysis as described earlier using the indicated antibodies. (C) Quantitative reverse-transcription polymerase chain reaction showing exogenous Iso1 or Iso2 transcript expression in CpA and CpD cells along with ACTB (housekeeping) compared with cell lines transfected with empty vector. Cycle threshold (Ct) expression values, rather than ΔΔCt ratios are presented, given the lack of RNF128 construct in the vector controls. Error bars represent the triplicate Ct range for each condition. (D) DDK-tagged UBCH5A (either WT or C85A mutant) and UBCH5C proteins were overexpressed in HEK293 cells, and 24 hours after transfection cell lysates were subjected to immunoprecipitation using FLAG-M2 beads. Thoroughly washed beads then were incubated with equal volumes of in vitro transcribed and translated V5-tagged either RNF128 Iso1 or Iso2. Unlike cells, Iso2 synthesis was approximately 1.9-fold higher compared with Iso1. Compared with input, 100-fold more amounts of Iso1 and Iso2 were used for interaction studies and the percentage binding was calculated as shown after normalization of input band intensity using ImageJ software (National Institutes of Health, Bethesda, MD). Lower panel: Expression of different UBCH5 family members in HEK293 cells. DDK, DYKDDDDK tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HC, Heavy Chain; IP, Immunoprecipitation; LC, Light Chain.