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. 2016 Jan 12;291(11):5817–5831. doi: 10.1074/jbc.M115.685917

FIGURE 9.

FIGURE 9.

Nectin-4-mediated enhancement of the prolactin-induced prolactin receptor tyrosine phosphorylation. A, no effect of nectin-1 on the prolactin-induced prolactin receptor tyrosine phosphorylation. A, panel a, HEK293E cells were co-transfected with various combinations of the indicated plasmids. The cells were serum-starved for 24 h and stimulated with 3 μg/ml prolactin for 10 min. The HA-tagged prolactin receptor (PRLR-HA) was immunoprecipitated with an anti-HA Ab, and the samples were subjected to Western blotting using the indicated Abs. Results are representative of three independent experiments. A, panel b, the band intensity of the phosphorylated PRLR-HA (pY PRLR-HA) was normalized to that of the total PRLR-HA, and the normalized value of the control FLAG-transfected cells stimulated with prolactin was set as 1.0. n.s., not significant. Error bars, S.E. B, nectin-4-mediated enhancement of the prolactin-induced prolactin receptor tyrosine phosphorylation. B, panel a, HEK293E cells were co-transfected with various combinations of the indicated plasmids. The cells were serum-starved for 24 h and stimulated with 3 μg/ml prolactin for 10 min. The HA-tagged prolactin receptor was immunoprecipitated with an anti-HA Ab, and the samples were subjected to Western blotting using the indicated Abs. Results are representative of three independent experiments. B, panel b, the band intensity of the phosphorylated PRLR-HA was normalized to that of the total PRLR-HA, and the normalized value of the control FLAG-transfected cells stimulated with prolactin was set as 1.0. n.s., not significant. *, p = 0.014 versus control as determined by Student's t test. Error bars, S.E. C, no enhancement of the prolactin receptor tyrosine phosphorylation when nectin-4 and the prolactin receptor were engaged in trans. C, panel a, preparation of the cells expressing FLAG alone, FLAG-nectin-4 alone, and both the PRLR-HA and GFP-JAK2. The cells were transfected with various combinations of the indicated plasmids, and the samples were subjected to Western blotting using the indicated Abs. C, panel b, no effect of nectin-4 on the prolactin-induced prolactin receptor tyrosine phosphorylation when both molecules were engaged in trans. HEK293E cells expressing FLAG-nectin-4 and HEK293E cells expressing PRLR-HA and GFP-JAK2 were co-cultured on a dish to induce trans-interaction between nectin-4 and the prolactin receptor. The cells were serum-starved for 24 h and stimulated with 3 μg/ml prolactin for 10 min. The PRLR-HA was immunoprecipitated with an anti-HA Ab, and the samples were subjected to Western blotting using the indicated Abs. Results are representative of three independent experiments. IP, immunoprecipitation; IB, immunoblotting; pY, phosphotyrosine.