Expression of antisense tTG construct in THP-1 cells reduces tTG content, association of tTG with integrins, and tTG expression on the cell surface.
THP-1 cells were transfected with pcDNA 3.1 vector alone or with the same vector containing a 1.1-kb antisense tTG construct,46to generate stable populations of THP-vector and THP-antisense transfectants. (A) The β1-, β2-, and β3-integrins and tTG were immunoprecipitated from 1 mg RIPA cell lysates of TPA-treated transfectants. The amounts of tTG in the immunoprecipitates were determined by SDS-PAGE followed by immunoblotting with anti-tTG mAb TG100. Arrowhead points to tTG bands. Note a reduction of the tTG content and the amounts of tTG complexed with β1-integrins and β3-integrins, in the THP-antisense transfectants. (B) The levels of cell surface tTG were determined by staining of live nonpermeabilized untreated and TPA-treated transfectants with polyclonal anti-tTG antibody, followed by fluorescein-conjugated anti–rabbit IgG and flow cytometry. The amounts of surface tTG were compared to that of untreated THP-vector cells, which was taken as 100%. ░, THP-vector; ■, THP-antisense. Shown are the means of triplicate determinations.
