Figure 2

Intracellular distribution intact and cleaved filamin A in BPH-1 cells. Cells were exposed to sterile 10B broth, 10⁹ CFU of U. parvum (UP), or cell culture supernatant (super) for 72 hours before examination by confocal microscopy (A), Western blot (B) and densitometry (C). Confocal images were taken at 600× magnification and the scale bar is equal to 10 μm. Cleaved and intact filamin A (Fil A) were stained with rabbit- anti C terminal filamin A (red). Intact Fil A was stained with mouse anti-filamin 1 (green). BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). Western blot analysis for the detection of cleaved filamin A was performed on cytosolic (cyt) and nuclear (nuc) fractions from uninfected (BPH) and infected (UP) cells. The black arrow is delineating GAPDH, which was used as a loading control and a confirmation that the nuclear fraction was not contaminated with cytosolic proteins. Quantitation of intact filamin A was performed by densitometry of the cytosolic fractions of uninfected and U. parvum infected cells. The average quantity within each blot was normalized by dividing the average quantity of filamin A protein band by the average quantity of the GAPDH band. Values represent the mean ± SD of 3 replicates from 3 independent experiments.