Journal: Nature Communications

Article Title: Lysophosphatidic acid activates Arf6 to promote the mesenchymal malignancy of renal cancer

doi: 10.1038/ncomms10656

Figure Lengend Snippet: ( a ) Binding of HA-EFA6B with Gα12 on LPA stimulation. EFA6B/shEFA6B cells (786-O/HA-EFA6B) were either stimulated with LPA or left untreated for 5 min, and the co-precipitation of endogenous trimeric G-proteins with HA-EFA6B was then analysed by anti-HA immunoprecipitation (IP) coupled with immunoblotting using the indicated antibodies. An irrelevant mouse IgG (IgG) was used as a control. ( b ) Endogenous association of Gα12 with EFA6B. Co-precipitation of Gα12, but not Gα13 or Gαq, with anti-EFA6B immunoprecipitants (IP) of 786-O cell lysates was examined, coupled with immunoblotting as indicated. ( c ) EFA6A-C, but not EFA6D, binds to GTP-Gα12. 293T cells were transfected with plasmids encoding HA-EFA6A-D or with an empty vector (Mock), together with plasmids encoding Gα12QL, Gα13QL, or GαqQL; and co-precipitation of these G-proteins with EFA6 family members was assessed as in a . ( d ) Schematic representation of the domain structure of EFA6B and its mutants. ( e ) The primary structures of the RGS domains of RhoGEFs, ARHGEF1, ARHGEF11, and ARHGEF12, and their amino acid alignment with the N-terminal regions of EFA6A-D. Conserved amino acids are marked in red boxes. ( f ) The RSG-like domain is essential for the binding to GTP-Gα12. Full-length HA-EFA6B (FL) and its mutants were expressed in 293T cells together with Gα12QL, as indicated, and their co-precipitation was assessed as in a . An empty vector (Mock) was included as a control. ( g , h ) Requirement for the RSG-like domain in the binding to endogenous Gα12 and the activation of Arf6. Full-length HA-EFA6B (FL) and its ΔRGS-like mutant were expressed in 786-O cells, in which endogenous EFA6B was silenced; and co-precipitation of HA-EFA6B proteins with Gα12 ( g ) and activation of Arf6 ( h ) were examined on LPA stimulation. ( a – c , f – h ) Total, total cell lysates.

Article Snippet: These cDNAs were ligated into the Not I site of pCX4-bsr , in which a synthesized HA fragment was inserted into the Bam HI/ Eco RI site. psd44-puro-based plasmids encoding constitutively active mutants of Gα12 (Gα12QL), Gα13 (Gα13QL) and Gαq (GαqQL) were purchased from Addgene (46825, 46828 and 46826, respectively).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Control, Transfection, Plasmid Preparation, Activation Assay, Mutagenesis

Journal:

Article Title: G Alpha-q/11 Protein Plays a Key Role in Insulin-Induced Glucose Transport in 3T3-L1 Adipocytes

doi:

Figure Lengend Snippet: Expression and effects of Gαq on glucose uptake into the 3T3-L1 adipocytes. (A) Differentiated 3T3-L1 adipocytes were infected with various concentrations (MOI = 1, 5, 10, and 20) of adenovirus expressing WT Gαq, Q209L-Gαq, or mock-infected control. After 60 h of infection, these cells were lysed, and total-cell lysates were analyzed by Western blotting with anti-Gαq/11 C-terminal antibody (top) or anti-Gβ1–5 antibody (bottom), as described in Materials and Methods. These experiments were repeated twice. (B) 3T3-L1 adipocytes were infected with various concentrations (MOI = 1, 5, 10, 20, 30, and 40) of adenovirus expressing WT Gαq, Q209L-Gαq, or mock control. After 60 h of infection, these cells were stimulated with 100 ng of insulin per ml for 1 h and 2-[3H]deoxyglucose uptake was measured as described in Materials and Methods. The data are the means and standard errors from four independent experiments.

Article Snippet: Horseradish peroxidase-linked anti-rabbit, anti-mouse, and anti-goat antibodies and anti-Akt1 and Gαq/11, antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Expressing, Infection, Western Blot

Journal:

Article Title: G Alpha-q/11 Protein Plays a Key Role in Insulin-Induced Glucose Transport in 3T3-L1 Adipocytes

doi:

Figure Lengend Snippet: Effects of anti-Gαq antibody or RGS2 protein microinjection on insulin-induced GLUT4 translocation in 3T3-L1 adipocytes. Serum-starved 3T3-L1 adipocytes on coverslips were incubated with or without insulin (4 and 10 ng/ml) for 20 min after the microinjection of sheep IgG, anti-Gαq/11 antibody, or RGS2 protein, mixed with sheep IgG as a marker, as described in Materials and Methods. (A) Representative cells of GLUT4 staining are shown in the top panels (a to d), and staining of injected RGS2/sheep IgG (c and d) or sheep IgG alone (a and b) is shown in the bottom panels. (B) Percentage of cells positive for GLUT4 translocation, calculated by counting at least 100 cells at each point. Injected materials are sheep IgG for Control, anti-Gαq/11 C-terminal antibody for Gq-Ab, and RGS2 protein mixed with sheep IgG for RGS2. The data are means and standard errors from four independent experiments.

Article Snippet: Horseradish peroxidase-linked anti-rabbit, anti-mouse, and anti-goat antibodies and anti-Akt1 and Gαq/11, antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Translocation Assay, Incubation, Marker, Staining, Injection

Journal:

Article Title: G Alpha-q/11 Protein Plays a Key Role in Insulin-Induced Glucose Transport in 3T3-L1 Adipocytes

doi:

Figure Lengend Snippet: Effects of Gαq expression on insulin-induced GLUT4 translocation in 3T3-L1 adipocytes. Serum-starved 3T3-L1 adipocytes on coverslips were incubated with or without insulin (10 ng/ml) for 20 min after 60 h of Gαq-expressing adenovirus infection (MOI = 10) (A) or after 24 h of nuclear microinjection with Gαq expression vector and with GFP expression vector as a marker (B). Fixed cells were stained with rabbit anti-GLUT4 antibody and incubated with TRITC-conjugated anti-rabbit IgG antibody, as described in Materials and Methods. The percentage of cells positive for GLUT4 translocation was calculated by counting at least 100 cells at each point. The data are means and standard errors from three independent experiments. (A) Cont., mock control adenovirus; Q209L, Q209L-Gαq-expressing adenovirus; WT, wild-type-Gαq-expressing adenovirus. (B) Control vector, GFP-expressing vector only.

Article Snippet: Horseradish peroxidase-linked anti-rabbit, anti-mouse, and anti-goat antibodies and anti-Akt1 and Gαq/11, antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Expressing, Translocation Assay, Incubation, Infection, Plasmid Preparation, Marker, Staining

Journal:

Article Title: G Alpha-q/11 Protein Plays a Key Role in Insulin-Induced Glucose Transport in 3T3-L1 Adipocytes

doi:

Figure Lengend Snippet: Microinjection and treatments of inhibitors and p110caax expression on Gαq expressed 3T3-L1 adipocytes. (A) After 60 h of Gαq-expressing adenovirus infection (MOI = 10), 3T3-L1 cells were injected with antiphosphotyrosine antibody (PY-20), GST-BARK, or sheep IgG as a control, prior to 3 h of insulin stimulation. Fixed cells were stained with rabbit anti-GLUT4 antibody and incubated with TRITC-conjugated anti-rabbit IgG antibody and AMCA-conjugated anti-sheep IgG antibody, as described in Materials and Methods. (B) After 60 h of Gαq-expressing adenovirus infection (MOI = 10) with or without p110caax-expressing adenovirus coinfection (CAAX), 3T3-L1 adipocytes were incubated with 300 nM wortmannin (Wort) or 0.1% DMSO vehicle for 60 min and with insulin (100 ng/ml) for 50 min or left untreated. 2-[3H]deoxyglucose uptake was measured as described in Materials and Methods. The data are means and standard errors from three independent experiments.

Article Snippet: Horseradish peroxidase-linked anti-rabbit, anti-mouse, and anti-goat antibodies and anti-Akt1 and Gαq/11, antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Expressing, Infection, Injection, Staining, Incubation

Journal:

Article Title: G Alpha-q/11 Protein Plays a Key Role in Insulin-Induced Glucose Transport in 3T3-L1 Adipocytes

doi:

Figure Lengend Snippet: Effect of Gαq antibody microinjection on GTPγS and p110caax signaling to GLUT4 translocation. 3T3-L1 cells infected with or without p110caax-expressing adenovirus were injected with anti-Gαq/11 antibody or sheep IgG with or without GTPγS. After 1 h, the cells were stimulated with insulin or left unstimulated, and then fixed. Fixed cells were stained with rabbit anti-GLUT4 antibody and incubated with TRITC-conjugated anti-rabbit IgG antibody and AMCA-conjugated anti-sheep IgG antibody as described in Materials and Methods.

Article Snippet: Horseradish peroxidase-linked anti-rabbit, anti-mouse, and anti-goat antibodies and anti-Akt1 and Gαq/11, antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Translocation Assay, Infection, Expressing, Injection, Staining, Incubation

Journal:

Article Title: G Alpha-q/11 Protein Plays a Key Role in Insulin-Induced Glucose Transport in 3T3-L1 Adipocytes

doi:

Figure Lengend Snippet: PI3-kinase activities of p110α and p110γ subunit in 3T3-L1 adipocytes after Gαq expression. Serum-starved 3T3-L1 adipocytes were incubated in the absence or presence of insulin (100 ng/ml) for 10 min or PDGF (50 nM) for 2 min, after 60 h of Gαq-expressing adenovirus infection. Cell lysates were immunoprecipitated (IP) with anti-p110α or anti-p110γ antibody, and immune complexes were assayed for their ability to phosphorylate phosphatidylinositol. Reaction products (phosphatidylinositol-3 phosphate) were analyzed by thin-layer chromatography, and signals were quantitated on a PhosphorImager, as described in Materials and Methods. The data are means and standard errors from three independent experiments. (A) p110α PI3-kinase activity; (B) p110γ PI3-kinase activity. Cont., control.

Article Snippet: Horseradish peroxidase-linked anti-rabbit, anti-mouse, and anti-goat antibodies and anti-Akt1 and Gαq/11, antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Expressing, Incubation, Infection, Immunoprecipitation, Thin Layer Chromatography, Activity Assay

Journal:

Article Title: G Alpha-q/11 Protein Plays a Key Role in Insulin-Induced Glucose Transport in 3T3-L1 Adipocytes

doi:

Figure Lengend Snippet: Role of PI3-kinase in insulin-induced GLUT4 translocation in Gαq expressed 3T3-L1 adipocytes. After 60 h of Gαq-expressing adenovirus infection (MOI = 10), 3T3-L1 cells were injected with anti-p110α, anti-p110γ, or sheep IgG as a control (Cont). After insulin stimulation, fixed cells were stained with rabbit anti-GLUT4 antibody and incubated with TRITC-conjugated anti-rabbit IgG antibody and FITC-conjugated anti-goat or anti-sheep IgG antibody, as described in Materials and Methods. The data are means and standard errors from three independent experiments.

Article Snippet: Horseradish peroxidase-linked anti-rabbit, anti-mouse, and anti-goat antibodies and anti-Akt1 and Gαq/11, antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Translocation Assay, Expressing, Infection, Injection, Staining, Incubation

Journal:

Article Title: G Alpha-q/11 Protein Plays a Key Role in Insulin-Induced Glucose Transport in 3T3-L1 Adipocytes

doi:

Figure Lengend Snippet: Effects of Gαq on insulin-induced membrane ruffling in 3T3-L1 adipocytes. 3T3-L1 cells were microinjected with anti-Gαq/11, anti-p110α antibody, RGS2 protein with sheep IgG, or sheep IgG as a control. After insulin stimulation, fixed cells were stained with rhodamine-phalloidin and FITC-conjugated anti-goat, anti-sheep, or anti-rabbit IgG antibody, as described in Materials and Methods. The data are means and standard errors from three independent experiments.

Article Snippet: Horseradish peroxidase-linked anti-rabbit, anti-mouse, and anti-goat antibodies and anti-Akt1 and Gαq/11, antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Staining

Journal:

Article Title: G Alpha-q/11 Protein Plays a Key Role in Insulin-Induced Glucose Transport in 3T3-L1 Adipocytes

doi:

Figure Lengend Snippet: Gαq activity and association of Gαq with insulin receptor β-subunit and p110α subunit of PI3-kinase. 3T3-L1 adipocytes were lysed and immunoprecipitated (IP) with anti-Gαq/11 or IR-β antibodies. Immunoprecipitates were analyzed by Western blotting with anti-p110α antibody (A, top), anti-Gαq/11 antibody (A, middle and bottom panel), or PY-20 antibody (B). These experiments were repeated twice.

Article Snippet: Horseradish peroxidase-linked anti-rabbit, anti-mouse, and anti-goat antibodies and anti-Akt1 and Gαq/11, antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Activity Assay, Immunoprecipitation, Western Blot

Journal:

Article Title: G Alpha-q/11 Protein Plays a Key Role in Insulin-Induced Glucose Transport in 3T3-L1 Adipocytes

doi:

Figure Lengend Snippet: Effects of Q209L-Gq on MAPK or p70S6-kinase activities in 3T3-L1 adipocytes. Serum-starved 3T3-L1 adipocytes were incubated in the absence or presence of insulin (100 ng/ml) for 10 min, after 60 h of Gαq-expressing adenovirus infection. Whole-cell lysates were subjected to SDS-PAGE and immunoblotted with phosphospecific MAPK antibody (A) or phosphospecific p70S6K antibody (B), as described in Materials and Methods. These experiments were repeated twice.

Article Snippet: Horseradish peroxidase-linked anti-rabbit, anti-mouse, and anti-goat antibodies and anti-Akt1 and Gαq/11, antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Incubation, Expressing, Infection, SDS Page

Journal:

Article Title: G Alpha-q/11 Protein Plays a Key Role in Insulin-Induced Glucose Transport in 3T3-L1 Adipocytes

doi:

Figure Lengend Snippet: Effects of anti-Akt or PKC-λ antibody injection on GLUT4 translocation in 3T3-L1 adipocytes. (A) After 60 h of Gαq-expressing adenovirus infection (MOI = 10), 3T3-L1 cells were injected with anti-Akt antibody (Akt-Ab), PKC-λ antibody (PKC λ-Ab), or sheep IgG as a control. After insulin stimulation, fixed cells were stained with rabbit anti-GLUT4 antibody and incubated with TRITC-conjugated anti-rabbit IgG antibody and FITC-conjugated anti-mouse or anti-sheep IgG antibody. The data are means and standard errors from three independent experiments. (B) Serum-starved 3T3-L1 adipocytes were incubated in the absence or presence of insulin (100 ng/ml) or PDGF (50 nM) for 10 min after 60 h of Gαq-expressing adenovirus infection. Whole-cell lysates were subjected to SDS-PAGE and immunoblotted with phosphospecific Akt antibody (top) or Akt antibody (bottom) for the gel shift assay, as described in Materials and Methods. These experiments were repeated twice.

Article Snippet: Horseradish peroxidase-linked anti-rabbit, anti-mouse, and anti-goat antibodies and anti-Akt1 and Gαq/11, antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Techniques: Injection, Translocation Assay, Expressing, Infection, Staining, Incubation, SDS Page, Electrophoretic Mobility Shift Assay