Figure 4

Intracellular distribution and quantification of calpastatin in uninfected, U. parvum infected, and supernatant treated BPH-1 cells. Cells were exposed to sterile 10B broth, 10⁹ CFU of U. parvum, or cell culture supernatant 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. Calpastatin was detected with rabbit polyclonal antibody (red). BPH-1 nuclei and U. parvum (white arrow) were identified with DAPI stain (blue). Western blot analysis for the detection of calpastatin was performed on cytosolic (cyt) and nuclear (nuc) fractions from uninfected cells (BPH), infected (UP), uninfected supernatant treated (BPH S) and infected supernatant treated (UP S) cells. M equals molecular weight marker. GAPDH was used as a loading control and a confirmation that the nuclear fraction was not contaminated with cytosolic proteins. Quantitation of calpastatin in cytosolic fractions was performed by densitometry. The average quantity within each blot was normalized by dividing the average quantity of calpastatin protein band by the average quantity of the GAPDH band within each blot. Values represent the mean ± SD of 2 biological replicates from 2 independent experiments.