Identification of new Cav1 pTyr14 interaction partners.

<p>(<b>a</b>) Pull-down of Cav1-pY14 interaction partners. Biotin-labeled Cav1 peptides either with (Cav1 pY14 peptide) or without (Cav1 peptide) phosphorylated Tyr14 were used as baits to precipitate interacting proteins from AGS cell lysates using a streptavidin-agarose pulldown assay. Precipitates were separated by SDS-PAGE and proteins visualized by silver staining. The proteins enriched in the Cav1 pY14 pulldown (indicated by numbers; see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003373#ppat-1003373-t001" target="_blank">table 1</a>) were identified by Maldi-MS/MS. (<b>b</b>) Western blot analysis of proteins precipitated from ME-180 cells using either biotin-labeled phosphorylated or non-phosphorylated Cav1 peptides as bait. PLCy1 as well as the regulatory p85 and catalytic p110 subunit of PI3K, are only detected in precipitates of the phosphorylated peptide with the respective primary antibodies. (<b>c</b>) PI3K-p85 interacts with Cav1 in N927 infected cells. Chang cells were infected with either N927 (PorB_IA, P⁻) or N138 (PorB_IB, P⁺) at an MOI 20 for 1 h or not infected (no inf). Endogenous Cav1 was precipitated and co-precipitated proteins were detected by Western blot with antibodies specific for Cav1 or PI3K-p85. (<b>D</b>) Relative amount of PI3 kinase quantified from (C). The bars represent the mean values ± SD of three independent experiments. (<b>E</b>) PI3 kinase recruitment to caveolin depends on activity of PLCy1. The experiment was performed as described in (C). 30 min prior to infection the PLCy1 inhibitor U73122 (10 µM) was added to the cells. (<b>F</b>) Relative amount of p85 quantified from three independent experiments. Shown are the mean values ± SD. (<b>G</b>) Interaction of Cav1 with Vav2 tested under conditions as described in (C). (<b>H</b>) Relative amount of Vav2 quantified from the experiment shown in (G). The bar graph shows the mean value of three independent experiments ± SD. p<0.05: *; p<0.01: **.