Figure 1

Transduction of HEK293T cells with Thap1 results in appearance of T1-LIR species of 27 kDa, 32 kDa, and 47 kDa. (a) HEK293T cells were transduced with adenovirus (Ad)-V5-Tagged human (h)Thap1 at MOI = 5. An aliquot (50 µg) of protein was analysed following separation in a 10% SDS-polyacrylamide gel, transfer, and immunoblotting with each of the following primary antibodies: Proteintech anti-Thap1 (far left panel); Santa Cruz anti-Thap1 antibody SC 3H3 (second panel from left); anti-V5 (third panel from left); NeuroMab anti-Thap1 (right panel). All 4 antibodies recognized a prominent species at 32 kDa (denoted as **) that was not present prior to transduction. Three of the antibodies (excluding the NeuroMab antibody) recognized a transduction-dependent species at 47 kDa (denoted by the arrow). A 29 kDa species (denoted by *) was detected by the Proteintech antibody before and after transduction. The absence of an increase in the intensity of the 29 kDa species recognized by the Proteintech antibody implies that this species is either wholly or partially attributable to a non-Thap1 cross-reacting species. Anti-V5 antibody recognized a species at 27 kDa (denoted by the arrowhead). (b) An aliquot (50 µg) of whole SDS protein extract or either nuclear (nuc) or cytoplasmic (cyt) fractions from HEK293T cells transduced with Ad-hThap1 or Ad-GFP control were separated and immunoblotted with either: (1) Proteintech anti-Thap1; (2) anti-GAPDH (cytoplasmic marker); or (3) anti-HDAC1 (nuclear compartment marker). Following fractionation, the 32 and 47 kDa T1-LIR species [symbols as in (a)] are primarily distributed to the nuclear fraction.