The real-time PCR results demonstrated that the gene expression l

The real-time PCR results demonstrated that the gene expression levels of 16 secretory learn more proteins exhibited the same trend of changes as the quantitative MS results (Figure 2A). Also, Western blot data showed that protein levels of six secretory proteins were significantly increased NVP-LDE225 solubility dmso in the CM and total cell

lysates after M. pneumoniae-infection, which were consistent with the proteomic results (Figure 2B). Therefore, from the RT-PCR and Western blot results, we found that these six secretory proteins (ADAM9, SERPINE1, IL-33, IGFBP4, Gal-1, MIF) were overexpressed in M. pneumoniae-infected A549 cells at mRNA and protein levels. Figure 2 Verification of up- or down-regulated proteins during M. pneumoniae infection. (A) RT-PCR analysis and quantitative analysis data of 16 secertory proteins during M. pneumoniae infection. compared to control (p < 0.05); data are presented as means ± SD. (B) Western blot analysis for 6 secretory proteins from total cell lysates and culture supernatants. Representative images were from three independent

experiments performed in duplicate. β-actin was used as internal control for total cell lysates. Cellular localization of the identified proteins The 256 identified proteins selleck chemicals llc were first categorized as classical secretory proteins or non-classical secretory proteins based on SingalP and SecretomeP analysis. Of the 256 proteins, 83 were categorized as classical secretory proteins and 69 as non-classical secretory proteins (see Additional file 5: Table S1). To determine Non-specific serine/threonine protein kinase whether some of the proteins could also be released via exosomes, the Exocarta exosome database were searched [22].

The results showed that among the proteins identified, 190 proteins were also listed in the exosomal protein database (see Additional file 5: Table S1). We next analyzed the ontology of the identified proteins based on cellular compartment. The results showed majority of the proteins belong to more than one GO class (Figure 3). Most of the proteins have a nuclear distribution (Figure 3A). Functional annotation clustering analysis by DAVID 6.7 showed that when considering only cellular compartment distribution, the proteins of the extracellular region, vesicle and extracellular matrix were over-represented (enrichment score (ES) of 12.24, 8.57, and 3.98, respectively) (Figure 3B). Similarly, the classification based on the cellular organelle of the differentially expressed proteins also showed that M. pneumoniae infection did not induce protein secretion from any specific cell organelle, but rather, altered the overall secretion of proteins from all the main organelles, including mitochondrion and lysosome (Figure 4 and see Additional file 7: Figure S4A). Enrichment in proteins residing in the extracellular region, especially extracellular matrix, extracellular space, and membrane-bound vesicle was observed (Figure 4 and see Additional file 7: Figure S4A). Moreover, when p value < 0.

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