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. 2018 Apr 11;9:713. doi: 10.3389/fmicb.2018.00713

FIGURE 2.

FIGURE 2

Expression of truncated MCV LT and small T (sT) antigens in CVG-1. (A) qRT-PCR analysis of T antigen transcripts in CVG-1 and three previously described MCV-positive MCC cell lines. The levels of total T antigen transcripts and sT transcript expressed in MCC cell lines were analyzed by the delta-delta Ct method to compare abundance of total T antigen (Left) and small T antigen transcripts (Right) relative to CVG-1 cells. Ct values from 18S rRNA amplification were utilized as an internal control. (B) Schematic representation of the LT truncation mutation in CVG-1, showing the ablation of the entire helicase domain. (C) Immunoblotting analysis of MCV LT and sT protein expression. MCV LT and sT protein expression was detected by CM2B4 and CM5E1 antibodies, respectively. CM2B4 detects full length LT (∼110 kDa) and multiply spliced 57kT (∼60 kDa) proteins in a positive control prepared from 293FT cells transfected with genomic MCV T antigen expression vector (TAg206), but not in a mock-transfected negative control. Cytokeratin 20 (CK20) expression was also detected as a marker of MCC. Hsp70 was used as a loading control. Asterisks indicate non-specific reactivities of CM2B4 antibody.