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. 2021 Jul 2;22(13):7169. doi: 10.3390/ijms22137169

Figure 3.

Figure 3

The ubiquitin conjugation lysine residue of MCPyV LT. (A) Potential LT ubiquitination lysine residues from 431 to 817aa were predicted using Rapid UBIquitination (RUBI), a sequence-based ubiquitination predictor. Four lysine residues in LT with ubiquitination probability over 0.3 are shown. Each lysine residue was substituted with arginine. (B) K585 is the ubiquitin conjugation site of MCPyV LT. HEK293 cells expressing either MCPyV LT protein or lysine mutants together with HA-Ub, as indicated, were immunoprecipitated with HA-conjugated agarose and immunoblotted for LT. Samples were loaded onto 8% SDS polyacrylamide gel. K585R mutant diminished its ability to interact with Ub. IgH = immunoglobulin heavy chain. (C) HEK 293 cells co-transfected with either Lt.wt or K585R mutant and HA-Ub were treated with MG132 (10 μM). HA-Ub proteins were immunoprecipitated with HA-conjugated agarose. Precipitated proteins were resolved using 4–20% Criterion TGX precast gradient gels (Bio-Rad) and analyzed by quantitative immunoblotting. IgH = immunoglobulin heavy chain, IgL = immunoglobulin light chain.