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Fig. S1A . The increase in the YFP signal at the GA after SDF1α stimulation was expressed as the translocation of Gβγ to the GA. ∗ p < 0.05 versus Gγ3. C , relative expression of YFP-Gγ9 at the PM, nucleus (nuc), and GA before and after SDF1α stimulation for 10 s in a representative DU145 cell based on the line scan analysis. D , the half time (t 1/2 ) of Gγ translocation from the PM to the GA after SDF1α stimulation. The images shown in A and C are representatives of five to eight experiments. The quantitative data are presented as means ± SD (n = 5–8). The scale bars represent 10 μm. GA, Golgi apparatus; PM, plasma membrane. " width="100%" height="100%">
Journal: The Journal of Biological Chemistry
Article Title: G protein βγ translocation to the Golgi apparatus activates MAPK via p110γ-p101 heterodimers
doi: 10.1016/j.jbc.2021.100325
Figure Lengend Snippet: Gβγ translocation from the PM to the GA in DU145, PC3, and HEK293 cells. A , Gγ9 translocation from the PM to the GA. The cells were cultured on 6-well dishes and transfected with YFP-Gγ9, Gβ1, Gαi1, and pmTurquoise2-Golgi (500 ng each). After starvation for 48 h, the cells were stimulated with SDF1α at 1 μg/ml. The images shown are obtained after stimulation for 10 s. B , quantification of the translocation of different Gγ subunits in complex with Gβ1 in response to SDF1α stimulation. The cells were transfected with individual YFP-Gγ, Gβ1, Gαi1, and pmTurquoise2-Golgi and stimulated as in A . The YFP signal at the GA before and after SDF1α stimulation was measured as shown in
Article Snippet: The YFP-tagged
Techniques: Translocation Assay, Cell Culture, Transfection, Expressing
Journal: The Journal of Biological Chemistry
Article Title: G protein βγ translocation to the Golgi apparatus activates MAPK via p110γ-p101 heterodimers
doi: 10.1016/j.jbc.2021.100325
Figure Lengend Snippet: Gγ9 subunit is required for ERK1/2 activation by SDF1α. A , SDF1α dose dependently activated ERK1/2. The cells were cultured on 6-well dishes. After starvation for 48 h, the cells were stimulated with different concentrations of SDF1α for 5 min. B , quantitative data shown in A . C , effect of PTX, gallein, and AMD3100 on ERK1/2 activation by SDF1α. The cells were incubated with PTX (100 ng/ml for 16 h), gallein (10 μM for 30 min), and AMD3100 (100 μM for 1 h) before SDF1α stimulation (200 ng/ml for 5 min). D , effect of Golgi-targeting GRK2ct (Golgi-GRK2ct) on ERK1/2 activation by SDF1α. The cells were transfected with Golgi-GRK2ct or its mutant GRK2ctR587Q and then stimulated with SDF1α. E , expression of endogenous Gγ3 and Gγ9 in CRISPR-Cas9–mediated Gγ9 ( left panel ) or Gγ3 ( right panel ) knockout cells by Western blotting using Gγ-specific antibodies. F , ERK1/2 activation in Gγ3 and Gγ9 knockout cells in response to SDF1α stimulation. G , rescue of ERK1/2 activation in response to SDF1α stimulation by transient expression of sgRNA-resistant Gγ9 (γ9res) in Gγ9 knockout PC3 cells. The quantitative data are presented as means ± SD (n = 3). The Western blots shown in each panel are representatives of at least three experiments. PTX, pertussis toxin.
Article Snippet: The YFP-tagged
Techniques: Activation Assay, Cell Culture, Incubation, Transfection, Mutagenesis, Expressing, CRISPR, Knock-Out, Western Blot
Journal: The Journal of Biological Chemistry
Article Title: G protein βγ translocation to the Golgi apparatus activates MAPK via p110γ-p101 heterodimers
doi: 10.1016/j.jbc.2021.100325
Figure Lengend Snippet: Inducible expression of Gβγ at the GA directly activates ERK1/2. A , inducible expression of Gγ2, Gγ3, and Gγ9 at the GA, but not at the PM, activated ERK1/2. The cells were cultured on 6-well dishes, transfected with individual FRB-Gγ and Gβ1, together with Golgi-Gγ or PM-Gγ (500 ng each), and then induced with rapamycin at 1 μM for 30 min. SDF1α simulation (200 ng/ml for 5 min) was used as a control. B , time courses of ERK1/2 activation by inducible expression of Gγ9 at the GA. C , quantitative data shown in B . D , effect of Golgi disruptors on Gγ9-mediated ERK1/2 activation. The cells transfected with FRB-Gγ9, Gβ1, and Golgi-FKBP were treated with BFA (3 μM), ilimaquinone (10 μM), monensin (5 μM), nigericin (2 μM), nocodazole (10 μM), and swainsonine (5 μM) for 40 min before incubation with rapamycin for 30 min to induce Gβγ translocation. E , dose-dependent action of BFA on Golgi-Gγ9-induced ERK1/2 activation. The cells were transfected with FRB-Gγ9, Gβ1, and Golgi-FKBP and then treated with BFA at different concentrations (0–4 μM) for 40 min before incubation with rapamycin for 30 min to induce Gβγ translocation. F , quantitative data shown in E . G , effect of Gβ, Gα, and GRK2ct on ERK1/2 activation by Golgi-Gγ9. The cells were transfected with FRB-Gγ9 and Golgi-FKBP with or without cotransfection with Gβ1, different Gα subunits, Golgi-GRK2ct, or GRK2ctR587Q (500 ng each) and then incubated with rapamycin for 30 min. ERK1/2 activation by SDF1α (200 ng/ml for 5 min) was used as a control. H , Gβγ translocation in the presence of Gα subunits. PC3 cells were transfected with venus-Gβ1, mCherry-Gαi1, FRB-Gγ9, and Golgi-FKBP (500 ng each) and then treated with rapamycin at 1 μM for 30 min. The quantitative data are presented as means ± SD (n = 3). The Western blots and images shown are representatives of at least three experiments. The scale bar represents 10 μm. BFA, brefeldin A; FRB, FKBP-rapamycin binding; GA, Golgi apparatus; PM, plasma membrane.
Article Snippet: The YFP-tagged
Techniques: Expressing, Cell Culture, Transfection, Activation Assay, Incubation, Translocation Assay, Cotransfection, Western Blot, Binding Assay
Journal: The Journal of Biological Chemistry
Article Title: G protein βγ translocation to the Golgi apparatus activates MAPK via p110γ-p101 heterodimers
doi: 10.1016/j.jbc.2021.100325
Figure Lengend Snippet: Depletion of p110γ and p101 abolishes ERK1/2 activation by SDF1α and Golgi-γ9. A , expression of p110γ in control and p110γ knockout cells. B , ERK1/2 activation by SDF1α at 200 ng/ml for 5 min in control and p110γ knockout cells. C , Golgi-γ9-induced ERK1/2 activation in p110γ knockout cells. The cells were transfected with FRB-γ9, Gβ1, and Golgi-FKBP (500 ng each) and then incubated with rapamycin for 30 min. D , ERK1/2 activation by SDF1α at 200 ng/ml for 5 min in p87 and p101 knockdown PC3 cells. E , Golgi-γ9–induced ERK1/2 activation in p87 and p101 knockdown PC3 cells. F , effect of Gγ3, Gγ9, and p110γ knockout on ERK1/2 activation by UK14304 at 1 μM for 5 min. G , effect of Gγ3, Gγ9, and p110γ knockout on ERK1/2 activation by EGF at 50 ng/ml for 5 min ( upper panel ), and insulin-like growth factor 1 at 200 ng/ml for 1 h ( lower panel ) in PC3 cells. The Western blots shown in each panel are representatives of at least three experiments.
Article Snippet: The YFP-tagged
Techniques: Activation Assay, Expressing, Knock-Out, Transfection, Incubation, Western Blot