Immunoprecipitated samples and supernatants left after immunoprecipitation were run under non-reducing (A) or reducing conditions (B) and analyzed by Western blot with anti-PRDX1 (A) or anti-HA (B) antibody. Supernatants were blocked with 40 mM NEM, concentrated and the virions immunoprecipitated with anti-influenza antibody. PRDX1 is not released associated with influenza virions.Ī549 cells were infected (+) or mock infected (-) as described in the Methods. Data are mean ± SE from three independent samples. Cell lysates were also analyzed after blocking with 40 mM NEM. A549 cells were infected with PR8 virus (+) or mock-infected (-) for 24 h as described in the Methods. Western blot analysis following non-reducing (12% for PRDXs and 15% for TXN1) SDS-PAGE of A549 supernatants and cell lysates. Influenza virus induces release of redoxins. The same blot was stripped and reprobed with anti-PRDX1 or anti-TXN1 antibody to locate the proteins (left, in both A and B). Proteins were then visualized by Western blot with streptavidin peroxidase. Immunoprecipitated proteins were run under non-reducing (two lanes on the left) or reducing conditions (the two lanes with DTT, on the right). Proteins in the NEM-blocked supernatants from BioGEE-pretreated, LPS-stimulated cells were immunoprecipitated with anti-PRDX1 (A) or anti-TXN1 (B). Proteins released in glutathionylated form. Please note that some residual PRDX1 was still detected after stripping (bottom gels). Arrows indicate the position of Actin and PRDX1. Bottom, the same membrane was stripped and reprobed with anti-actin as a reference intracellular protein. Western blot analysis following non-reducing SDS-PAGE (12% acrylamide) of RAW264.7 supernatants obtained from cells cultured with or without 100 ng/ml LPS for 24 h (1 x10 6 cells in 6 well plates in 1ml of OPTI-MEM) and of different percentages of cell lysate obtained from the same number of cells, cultured without LPS, resuspended in 1ml sample buffer (to mimic a supernatant containing the maximum amount of PRDX1 that would be released if 5, 10, 20, 40 or 100% of the cells were necrotic). Cell lysates were also analyzed after blocking with NEM.Ĭell lysis cannot account for LPS-induced release of PRDX1. Supernatants were blocked with 40 mM NEM immediately after collection, to prevent thiol-disulfide exchange. Western blot analysis following non-reducing (A) or reducing (B) SDS-PAGE (10% acrylamide for VIM, 12% for PRDXs, 15% for TXN1 and PFN1) of RAW264.7 supernatants cultured with and without 100 ng/ml LPS for 24 h. LPS induces release of PRDX1, PRDX2, TXN1, VIM and PFN1. Proteins identified from BioGEE-treated samples.
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