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. 2013 Jul 3;288(32):23252–23263. doi: 10.1074/jbc.M113.452037

FIGURE 1.

FIGURE 1.

Phosphorylation of SHP at Thr-55 in response to BA or FGF19 signaling. A, tandem mass (MS/MS) spectrum of the SHP peptide showing phosphorylation of Thr-55 (T∧, phosphorylated Thr-55; T#, Thr-55 with loss of water; R*, methylated Arg-57). FLAG-SHP expressed in HepG2 cells treated with CDCA was isolated, visualized by colloidal staining, and excised for MS/MS analysis. B and C, phospho-Thr levels of FLAG-SHP in HepG2 cells treated with FGF19, CDCA, or GW4064 for 1 h were detected by IP of FLAG-SHP followed by IB using phospho-Thr antibody. D and E, phospho-Thr levels of endogenous SHP in mouse liver or HepG2 cells treated with FGF19 or CDCA are shown. In B and E, bands were quantified and phosphor-Thr SHP levels relative to untreated cells (CTL) shown (right panel) and statistical significance was determined by the Student's t test, * indicates p < 0.05, S.E., n = 3. F, FLAG-SHP WT or T55A was expressed in HepG2 cells and phospho-Thr SHP was detected by IP/IB analysis. G and H, the experimental outline is shown at the top. Phospho-Thr-55 of SHP in liver extracts was detected by IP/IB analysis using general phosphor-Thr antibody (G) or phosphor-Thr-55-specific SHP antibody (H). FLAG-SHP and control GFP from the adenoviral infection are shown at the bottom. I, phosphorylation of endogenous SHP at Thr-55 in mouse liver extracts was detected by IB using a phosphor-Thr-55-specific SHP antibody. J, alignment of the SHP region containing Thr-55 (underlined) from various species.