Figure 3
Figure 3. S49 of RGS18 contributes to binding of 14-3-3 to RGS18. (A) Evaluation of the RGS18 and 14-3-3 interaction in co-immunoprecipitation studies. The FLAG-RGS18 constructs (wt-RGS18, S49A-RGS18, S216A-RGS18, S216E-RGS18, S218A-RGS18, and S49A/S218A RGS18) were coexpressed with the myc-14-3-3γ construct in HEK293T cells, lysed, and immunoprecipitated using anti-FLAG–coupled beads. After SDS-PAGE, the Western blots of IPs were incubated with rabbit anti-myc Ab (top panel, 14-3-3) and reincubated with anti-FLAG Ab as a loading control (middle panel, RGS18). Lysate Western blots were incubated with anti-myc Ab as a loading control (bottom panel, total 14-3-3). Shown is a representative blot of independent experiments performed 7 times. A summary of the results obtained is shown in the densitometry panel. (B) Blots of experiments shown in panel A were analyzed by densitometry and data are expressed as means ± SEM representing 7 independent experiments. Statistical significance of relative 14-3-3 binding in relation to wt-RGS18 was detected using 1-way ANOVA in combination with the Tukey posttest (wt to S49A, **P < .01; wt to S216E, S218A, and S218A/49A, ****P < .0001). (C) Phos-tag analysis of overexpressed RGS18. FLAG-RGS18 constructs wt-RGS18, S49A-RGS18, S216A-RGS18, S218A-RGS18, and S49A/S218A RGS18 were expressed in HEK293T cells. Cells were lysed and subjected to Phos-tag–supplemented SDS-PAGE and Western blotting. An aliquot of wt-RGS18 lysate was incubated with λ phosphatase for 1 hour at 30°C before SDS-PAGE and Western blot analysis. Blots were then incubated with mouse anti-RGS18 Ab. Appearing bands were labeled as b, s1, s2, or s3 with increasing apparent molecular weight. (D) Phos-tag analysis of endogenous RGS18. Washed platelets were either unstimulated or incubated with forskolin (10μM, 30 minutes) or thrombin (0.1 U/mL, 30 seconds) before lysis and Phos-tag–supplemented SDS-PAGE and Western blots. An aliquot of nonstimulated platelet lysate was incubated with λ phosphatase for 1 hour at 30°C before SDS-PAGE and Western blot analysis. Blots were then incubated with mouse anti-RGS18 Ab. For appearance of band b in phosphatase-treated samples, a 30-minute exposure is shown (first lane), which is separated by a black line from a shorter exposure (5 minutes) of lanes 2 through 4 of the same Western blot. Blots shown in panels C and D are representative of 4 independent experiments.

S49 of RGS18 contributes to binding of 14-3-3 to RGS18. (A) Evaluation of the RGS18 and 14-3-3 interaction in co-immunoprecipitation studies. The FLAG-RGS18 constructs (wt-RGS18, S49A-RGS18, S216A-RGS18, S216E-RGS18, S218A-RGS18, and S49A/S218A RGS18) were coexpressed with the myc-14-3-3γ construct in HEK293T cells, lysed, and immunoprecipitated using anti-FLAG–coupled beads. After SDS-PAGE, the Western blots of IPs were incubated with rabbit anti-myc Ab (top panel, 14-3-3) and reincubated with anti-FLAG Ab as a loading control (middle panel, RGS18). Lysate Western blots were incubated with anti-myc Ab as a loading control (bottom panel, total 14-3-3). Shown is a representative blot of independent experiments performed 7 times. A summary of the results obtained is shown in the densitometry panel. (B) Blots of experiments shown in panel A were analyzed by densitometry and data are expressed as means ± SEM representing 7 independent experiments. Statistical significance of relative 14-3-3 binding in relation to wt-RGS18 was detected using 1-way ANOVA in combination with the Tukey posttest (wt to S49A, **P < .01; wt to S216E, S218A, and S218A/49A, ****P < .0001). (C) Phos-tag analysis of overexpressed RGS18. FLAG-RGS18 constructs wt-RGS18, S49A-RGS18, S216A-RGS18, S218A-RGS18, and S49A/S218A RGS18 were expressed in HEK293T cells. Cells were lysed and subjected to Phos-tag–supplemented SDS-PAGE and Western blotting. An aliquot of wt-RGS18 lysate was incubated with λ phosphatase for 1 hour at 30°C before SDS-PAGE and Western blot analysis. Blots were then incubated with mouse anti-RGS18 Ab. Appearing bands were labeled as b, s1, s2, or s3 with increasing apparent molecular weight. (D) Phos-tag analysis of endogenous RGS18. Washed platelets were either unstimulated or incubated with forskolin (10μM, 30 minutes) or thrombin (0.1 U/mL, 30 seconds) before lysis and Phos-tag–supplemented SDS-PAGE and Western blots. An aliquot of nonstimulated platelet lysate was incubated with λ phosphatase for 1 hour at 30°C before SDS-PAGE and Western blot analysis. Blots were then incubated with mouse anti-RGS18 Ab. For appearance of band b in phosphatase-treated samples, a 30-minute exposure is shown (first lane), which is separated by a black line from a shorter exposure (5 minutes) of lanes 2 through 4 of the same Western blot. Blots shown in panels C and D are representative of 4 independent experiments.

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