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Figure 2 | Molecular Cancer

Figure 2

From: Deletion or insertion in the first immunoglobulin-plexin-transcription (IPT) domain differentially regulates expression and tumorigenic activities of RON receptor Tyrosine Kinase

Figure 2

Expression, localization, and phosphorylation of RON160 and RONE5/6inin MDCK cells. A) Expression of RON160 and RONE5/6in in MDCK cells. Cell lysates (50 μg/sample) from M-RON, M-RON160, or M-RONE5/6in after 72 h incubation were analyzed by Western blot analysis using rabbit IgG antibody to RON C-terminus [29]. β-actin was used as the loading control. Both pro- and mature proteins were observed. The presence of RONp110 in M-RONE5/6in cells was indicated. B) Immunofluorescent analysis of RON, RON160, and RONE5/6in expression on cell surface. Cells (1 × 106 cells per sample) were incubated with 1 μg/ml of Zt/g4 specific to the RON extracellular domain [30]. Goat-anti-mouse IgG coupled with FITC was used as the secondary antibody. MDCK cells were used as the negative control. Fluorescent intensity of individual samples was analyzed by FACScan. C) Spontaneous and MSP-induced phosphorylation of RON, RON160, and RONE5/6in in MDCK cells. Cells (3 × 106 cells/sample) after 72 h incubation were stimulated at 37°C with or without 2 nM of MSP for 10 min. Cellular proteins (250 μg/sample) were immunoprecipitated with Zt/g4 (1 μg/ml) followed by Western blot analysis using anti-phosphotyrosine mAb PY-100. Membranes were also reprobed with rabbit anti-RON C-terminus antibodies as the loading control. D) Effect of MSP on RON, RON160, and RONE5/6in-mediated phosphorylation of downstream signaling proteins. Cells were stimulated for 10 min with MSP as described in C. Western blot analysis using antibodies to regular or phosphorylated Erk1/2 and AKT were carried out as previously described [29]. Data shown here are from one of three experiments with similar results.

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