Interaction of the SSTR5 with SNX27 and NHERF1.

A. Cells were transfected with expression vectors coding for mRFP-tagged wt SSTR5 or the truncation mutant SSTR5ΔCT, and a GFP-fusion of SNX27. After cell lysis, receptors were precipitated using RFP-trap matrix. Input and precipitate (IP) samples were analyzed by Western Blot using mRFP and SNX27 specific antibodies. The positions of endogenous and GFP-tagged recombinant SNX27 are indicated. B. A GFP-fusion of NHERF1 was coexpressed with either of the two receptor variants. After cell lysis, receptors were precipitated using RFP-trap matrix. Input and precipitate (IP) samples were analyzed by Western Blot using mRFP and GFP specific antibodies. C. mRFP-tagged SSTR5 was coexpressed with GFP-tagged PDZ proteins; cells lysates were subjected to immunoprecipitation using GFP-trap matrix. Input and precipitate samples were analyzed by Western blotting. For A–C, a typical of three experiments is shown in each case. D. GFP-tagged PDZ domain proteins from cell lysates of transfected 293 cells (input) were precipitated with control NHS-sepharose, or sepharose conjugated with the SSTR5 C-terminal peptide carrying the PDZ ligand sequence. Input and precipitate samples were analyzed by Western blotting using anti-GFP antibody. For quantitation, the ratio of precipitate to input signal intensity was determined. *,#, significantly different from PIST and NHERF1, respectively (p<0.05; Anova (p = 0.015), followed by t-test with Bonferroni correction; n = 4).