All comparisons were two tailed; P<0.05 was considered as significant. Data were analyzed using spss 15. During the course of our experiments with SH-SY5Y neuroblastoma cells, we detected, using the EZ-PCR method, a mycoplasma contaminating our cell culture. The mycoplasma was identified as M. hyorhinis and designated as a neuroblastoma-derived M. hyorhinis (NDMH) strain. SH-SY5Y cells in the GM were infected with NDMH. NDMH-infected and noninfected (clean) cells were induced to differentiate, STA-9090 in vivo as described in Materials and methods. The morphology of the differentiated infected cells was similar to that of the clean cells,
with mycoplasma observed around the cells and in the medium of the infected cultures (Fig. 1a). PCR was carried out to determine the presence and absence of mycoplasmas in the cultured cells (Fig. 1b). Calpastatin in the control and in the NDMH-infected cells was analyzed by immunoblotting, as described in Materials and
methods. Calpastatin levels were significantly higher in the infected cells than in the clean cells, with levels of 208±20.3% (n=4), compared with the calpastatin levels in the clean cells (Fig. 2a and b). As can be seen in Fig. 2a, using a polyclonal anticalpastatin antibody, a major band of about 110 kDa was identified in both the clean and the infected cells. The NDMH did not exhibit the 110 kDa band, with a band of approximately 70 kDa observed (Fig. 2a). Using a monoclonal calpastatin antibody specific
for human calpastatin, the 110 kDa band was observed selleck kinase inhibitor in the clean and infected cells, whereas no bands were observed in the mycoplasma extracts, confirming that the mycoplasma did not have any human calpastatin (Fig. 2c and d). The results indicate that the high levels of calpastatin in the infected cells do not originate in the mycoplasma. Immunoblotting was also carried out for the identification of calpain. μ-Calpain was identified as a band of approximately 80 kDa, present in the clean and in the infected cells. It was not present in the NDMH (Fig. 3a). The μ-calpain levels in the infected cells were 139±9.7% (n=3), as compared with the levels in the clean cells (Fig. 3b). Calpain activation is generally considered to be associated with autolysis, with the 80 kDa subunit level indicating inactive calpain L-gulonolactone oxidase (Niapour et al., 2008), as is the case with inactive procaspases. The results thus suggested that calpain was less active in the infected cells than in the clean cells. In order to determine whether the higher calpastatin levels in the infected cells, observed by immunoblotting, interfere with calpain activity, casein zymography was carried out, as described in Materials and methods. Zymography allows the electrophoretic separation of calpastatin from calpain and estimation of calpain caseinolytic activity directly on the gel, without inhibition by the usual calpastatin content of the cell (Raser et al., 1995).