FIG. 3.
Expression of Ehformin-1 and -2 in stable transformants of E. histolytica. (A) Western blot analysis of the expression of recombinant Ehformin-1 and -2 in stable transformants of E. histolytica. Cell lysates from control (lane 1), HA-tagged Ehformin-1 (lane 2), and His-HA-tagged Ehformin-2 (lane 3) transformants were separated by 7% SDS-PAGE and transferred by Western blotting (WB). Western blots were hybridized with a monoclonal anti-HA antibody to detect the epitope-tagged Ehformin-1 and -2. A similar blot was hybridized with a polyclonal anti- E. histolytica β-tubulin antibody as a loading control. (B) Quantitative RT-PCR analysis of E. histolytica formins in the respective transformants. Real-time PCR was performed on cDNA prepared from control and forminEh transformants (x axis) using specific forminEh primer sets. ActinEh primers were used as an endogenous control. The relative expression of Ehformin-1 and Ehformin-2 in control, Ehformin-1, and Ehformin-2 transformants was plotted. Error bars, standard deviations (n = 3). (C) Association of Ehformin-1 and -2 with the actin-rich subcellular fraction. Crude lysates (lanes 1 and 2) and the actin-rich fraction (lanes 3 and 4) from Ehformin-1 (lanes 1 and 3) and Ehformin-2 (lanes 2 and 4) transformants were separated by SDS-PAGE, transferred to Western blots, and hybridized with monoclonal anti-HA and anti-β-actin antibodies. M, molecular sizes. Arrow indicates bands for HA-tagged Ehformin-1 and HA-tagged Ehformin-2.