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. 2009 Sep 25;5(9):e1000595. doi: 10.1371/journal.ppat.1000595

Figure 2. AvrA is phosphorylated when expressed in S. cerevisiae or in mammalian cells.

Figure 2

A AvrA is phosphorylated in S. cerevisiae. Extracts of S. cerevisiae expressing the indicated FLAG epitope tagged alleles of AvrA or YopJ were analyzed by western immunoblot with antibodies directed to the FLAG epitope. The lower panel shows longer exposure of the gel to visualize the dually phosphorylated form of AvrA (see text for details). B Transiently expressed AvrA is phosphorylated in mammalian cells. COS cells were transfected with plasmid expressing FLAG epitope tagged AvrA, YopJ, or their catalytic mutants AvrAC172S and YopJC172S. Twenty-four hours after transfection, cell extracts were examined by western immunoblot with an antibody directed to the FLAG epitope. C The mobility shift of transiently expressed AvrA is reversed after phosphatase treatment. COS cells were transfected with plasmid expressing FLAG epitope tagged AvrA and twenty-four hours after transfection, cell extracts were treated with lambda phosphatase in the presence or absence of a phosphatase inhibitor and examined by western immunoblot as indicated above. D AvrA is phosphorylated and persists within mammalian cells after its delivery via the S. Typhimurium TTSS. Henle-407 cells were infected with a S. Typhimurium strain expressing FLAG epitope-tagged AvrA and at the indicated times after infection, the presence of AvrA in the translocated and bacteria-associated protein fractions was probed by western immunoblot with an anti FLAG antibody. The lower panels show a longer exposure of the gel to visualize the dually phosphorylated form of AvrA. E Ser3 and Ser14 are essential for AvrA phosphorylation in mammalian cells. Henle-407 cells were infected with a S. Typhimurium strain expressing FLAG epitope-tagged AvrA or its mutant AvrAS3C S14C and at the indicated times after infection, the presence of AvrA in the translocated and bacteria-associated protein fractions was probed by western immunoblot with an anti FLAG antibody. The lower panels show a longer exposure of the gel to visualize the dually phosphorylated form of AvrA.