Polyphosphate Maintains Intestinal Homeostasis Figure 7. Poly P interacts with integrin b1 and activates p38 MAPK pathway. (A) Western blot analysis of p38, ERK, JNK and Akt phosphorylation in Caco2/BBE by 10 mg/mL of poly P. (B) Expression and localization of integrin b1 and b3 in mouse small intestine. Immunohistochemistry for integrin b1 and b3 were performed by using anti-integrin b1, rabbit-poly antibody and anti-integrin b3, rabbit-poly antibody. (C) Inhibition of poly P-induced p38 MAPK phosphorylation by RGD peptide. C57Bl/6 mice small intestinal loops were filled with RPMI 1640 medium containing 10 mg/mL of poly P, 10 mM of Gly-Arg-Gly-Asp-Ser (GRGDS) or Ser-Asp-Gly-Arg-Gly (SDGRG) 2 h at 37uC in a 5% CO2 incubator. Their p38 MAPK phosphorylation was detected by western blot (n = 3, each group). (D) Poly P-integrin b1 or b3 interaction analysis. Reaction mixture of 32P-labeled poly P and integrin a3b1 or aVb3 was immunoprecipitated with integrin b1 or b3 antibody and protein G beads. Immunoprecipitated 32P-labeled poly P was determined with liquid scintillation counting. doi:10.1371/journal.pone.0023278.g007 taa-39, anti-sense, 59-gataaggcttggcaacccaagtaa-39 for the analysis of mouse IL-10, sense, 59-tgtcttagccagtcccgaaacc-39, anti-sense, 59- tcttcatgatcgatgtcttcagcag-39 for the analysis of mouse IL-12, and sense, 59-tctcgaatgtaccaggagccatatc-39, anti-sense,59-agcaccttg- gaagccctacaga-39 for the analysis of mouse IL-4) in triplicate. The averaged mRNA expression of these cytokines was normal- PLoS ONE | www.plosone.org 11 August 2011 | Volume 6 | Issue 8 | e23278