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. Author manuscript; available in PMC: 2014 Aug 15.
Published in final edited form as: Cell Host Microbe. 2013 Aug 14;14(2):125–135. doi: 10.1016/j.chom.2013.06.008

Figure 6. MCV small T antigen exon1A LSD domain is required for sT-induced transformation in a PP2A-independent manor.

Figure 6

(A) LSD domain is critical for sT-induced transformation. Rat-1 cells were stably transduced with vector, wild type sT, mutants (sT.LSD91-95A, L142A), and SV40 sT. Both wild-type MCV sT and sT.L142A reproducibly formed colonies after 3 weeks of growth in soft agar, whereas the MCV sT.LSD91-95A mutation ablated transforming activity. Cells were stained with crystal violet (upper). Transformation-associated foci were photographed (×40) (middle) and colony number per high-power field was counted (bottom). All assays were performed in triplicate. Error bars represent SEM; n = 3. (B) All sT constructs show similar levels of expression in Rat-1 cell lines from transformation assays. Stable sT expressing cell lines used for soft agar assay were tested by immunoblotting with mixed antibodies, CM8E6 and pAb419 for MCV and SV40 sT detection, respectively. (C) LSD mutation does not affect sT binding to PP2A. Immunoprecipitation analysis was performed to measure PP2A binding capacity of sT mutants stably expressed in Rat-1. Either MCV sT antibody (CM8E6) or SV40 sT antibody (pAb419) was used for immunoprecipitation of sT and endogenous PP2A was detected. The non-transforming LSD mutant sT interacts with PP2A, similar to wild type sT. A PP2A binding mutant L142A ablates its binding to PP2A but retains its transforming activity (Figure 6A). These data indicate that the sT transforming activity is PP2A-independent. SV40 sT was used as a positive control of PP2A binding and a negative control for the transformation. See also Figure S5.