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. Author manuscript; available in PMC: 2009 Apr 1.
Published in final edited form as: Mol Immunol. 2007 Dec 21;45(7):1825–1836. doi: 10.1016/j.molimm.2007.11.002

Figure 3.

Figure 3

LscCT expression activates endogenous Lsc RGS and RhoGEF activity. A) A20 stable transfectants expresssing either YFP or CFP alone as controls, full-lenth Lsc (Lsc-FLAG) or LscCT (LscCT-CFP) were stimulated with 20 µM LPA for the indicated time in minutes and phosphorylated ERK1/2 and total ERK2 measured by Western blot analysis. Numbers below blots indicate relative expression of pERK1/2 or ERK2 in Lsc tranfectants compared to controls. B) Level of phosphorylated PRK1/2 (pPRK1/2) and total PRK1 in cell lysates from A20 parental cells and tranfectants expressing CFP (control transfectants), Lsc-FLAG or LscCT. C) Stable A20 cell transfectants expressing LscCT harbor significantly more active RhoA compared to control transfectants relative to the amount in Lsc-FLAG transfectants and as measured by a GTP-RhoA specific ELISA. Data in panels A, B, and C are representative of 4 experiments from at least two independent stable transfectants of each transgene. D) 293 cells transiently expressing either RhoA-YFP alone or RhoA-YFP together with either LscCT or full length Lsc or both were evaluated for level of RhoA, LscCT and Lsc by Western analysis (bottom) or level of GTP-RhoA by ELISA (top). GTP-RhoA measurement by ELISA in (D) includes data from 3 independent transient transfections. A20 parental cells and transfectants were serum-starved for 3–12 hours prior to cell harvest and evaluating RhoA-GTP, PRK1/2 phosphorylation, and total PRK1 levels.