two-way anova with bonferroni post tests and densitometric analyses  (GraphPad Software Inc)

Journal: The EMBO Journal

Article Title: Sub-picomolar relaxin signalling by a pre-assembled RXFP1, AKAP79, AC2, β-arrestin 2, PDE4D3 complex

doi: 10.1038/emboj.2010.168

Figure Lengend Snippet: A sub-picomolar relaxin response. cAMP biosensors in single cells revealed a response to sub-picomolar concentrations of relaxin ( n =10–74). ( A ) Stimulation of HEK293 cells expressing pmEpac2 or co-expressing pmEpac2 and RXFP1 with a sub-maximal concentration of relaxin (1 nM) over 5 min. ( B ) HEK293 cells co-expressing RXFP1 and pmEpac2 were stimulated with vehicle (0.001% trifluoroacetic acid, TFA), or a low (1 pM), sub-maximal (1 nM) or high (100 nM) concentration of relaxin over 5 min. All cells were stimulated at 0 min, a maximal cAMP response (FIP; 10 μM forskolin, 100 μM IBMX, 100 nM PGE 1 ) was induced at 6 min. Note that saturation of the probes is not achieved with relaxin stimulation, as FIP gives a greater response. ( C ) HEK293 cells expressing pmEpac2. ( D ) Relaxin (10 aM–100 μM) stimulation of RXFP1 co-expressed with pmEpac2 or pmEpac2 R297E (a sensor that cannot bind cAMP) in HEK293 cells. ( E ) Relaxin (1 pM–1 μM) or INSL3 (100 aM–100 μM) stimulation of RXFP2 co-expressed with pmEpac2 in HEK293 cells. ( F ) HEK293 cells expressing pmEpac2. ( G ) Relaxin (10 aM–100 μM) stimulation of HeLa cells (endogenously express RXFP1) transfected with glEpac2, in the presence of forskolin (1 μM) and the Gα i/o inhibitor pertussis toxin (PTX; 100 ng/ml, 16 h). ( H ) Relaxin (10 aM–100 μM) stimulation of primary rat cardiac fibroblasts (endogenously express RXFP1) infected with Ad-glEpac1, in the presence of forskolin (1 μM) and PTX. ( I ) HeLa cells (top) and rat cardiac fibroblasts (bottom) expressing glEpac2 or Ad-glEpac1, respectively. Data are expressed as the 5 or 12 min area under the curve (AUC). Symbols represent means, error bars, s.e.m. * P <0.05, ** P <0.01 and *** P <0.001 relaxin versus basal; ^ P <0.05, ^^ P <0.01 and ^^^ P <0.001 INSL3 or relaxin with PTX versus basal; two-way ANOVA with Bonferroni post tests.

Article Snippet: Single-cell FRET data were analysed by two-way ANOVA with Bonferroni post tests and densitometric analyses, by one-way ANOVA with Newman–Keuls multiple comparison test (GraphPad Prism).

Techniques: Expressing, Concentration Assay, Transfection, Infection

Journal: The EMBO Journal

Article Title: Sub-picomolar relaxin signalling by a pre-assembled RXFP1, AKAP79, AC2, β-arrestin 2, PDE4D3 complex

doi: 10.1038/emboj.2010.168

Figure Lengend Snippet: A sub-picomolar relaxin response requires Gα s and Gβγ. Sub-picomolar relaxin signalling was examined at the G-protein level in HEK293 cells co-expressing RXFP1 and pmEpac2, and stimulated with vehicle (0.001% TFA), 10 fM or 10 nM relaxin ( n =43–51). ( A ) Cells were pre-incubated with the Gα i/o inhibitor PTX (100 ng/ml, 16 h) and stimulated with 10 fM relaxin. ( B ) The 5 min area under the curve (AUC) from ( A ) for TFA, 10 fM and 10 nM relaxin. ( C ) Cells were pre-incubated with the Gα s -specific antagonist NF449 (10 μM, 30 min), or the Gα s -activator cholera toxin (200 ng/ml, 90 min) and stimulated with 10 fM relaxin. ( D ) The 5 min AUC from ( C ) for TFA, 10 fM and 10 nM relaxin. ( E ) Gβγ subunits were inhibited by gallein (10 μM, 30 min) or mSIRK (5 μM, 15 min) and stimulated with 10 fM relaxin. ( F ) The 5 min AUC from ( E ) for TFA, 10 fM and 10 nM relaxin. Data are expressed relative to the maximal cAMP response (FIP; 10 μM forskolin, 100 μM IBMX, 100 nM PGE 1 ), or as the 5 min AUC. Bars represent means, error bars s.e.m. *** P <0.001 versus own basal; ^ P <0.05, ^^ P <0.01 and ^^^ P <0.001 versus response to relaxin alone; two-way ANOVA with Bonferroni post tests.

Article Snippet: Single-cell FRET data were analysed by two-way ANOVA with Bonferroni post tests and densitometric analyses, by one-way ANOVA with Newman–Keuls multiple comparison test (GraphPad Prism).

Techniques: Expressing, Incubation

Journal: The EMBO Journal

Article Title: Sub-picomolar relaxin signalling by a pre-assembled RXFP1, AKAP79, AC2, β-arrestin 2, PDE4D3 complex

doi: 10.1038/emboj.2010.168

Figure Lengend Snippet: A cellular response to sub-picomolar relaxin requires AC2 and an AKAP. Sub-picomolar relaxin signalling was examined downstream of Gα s and Gβγ, in HEK293 co-expressing RXFP1 and pmEpac2, and stimulated with vehicle (0.001% TFA), 10 fM or 10 nM relaxin ( n =11–52). ( A ) AC activity was inhibited by 2′, 5′-dd′-3′-AMP-bis( t -BuSATE) (ddAB; 1 μM, 30 min) and cells were stimulated with 10 fM relaxin. ( B ) The 5 min area under the curve (AUC) from ( A ) for TFA, 10 fM and 10 nM relaxin. ( C ) The effect of overexpression of AC2 or AC7 on the response to 10 fM relaxin. ( D ) The 5 min AUC from ( C ) for TFA, 10 fM and 10 nM relaxin. ( E ) The effect of AC2 overexpression with and without co-expression of RXFP1 upon basal cAMP. ( F ) The 5 min AUC from ( E ) for TFA, 10 fM and 10 nM relaxin. ( G ) AKAP/PKA binding was inhibited using St-Ht31 or control peptide St-Ht31-P (both 20 μM, 45 min) and cells were stimulated with 10 fM relaxin. ( H ) The 5 min AUC from ( G ) for TFA, 10 fM and 10 nM relaxin. Data are expressed relative to the maximal cAMP response (FIP; 10 μM forskolin, 100 μM IBMX, 100 nM PGE 1 ), or as the 5 min AUC. Bars represent means, error bars s.e.m. * P <0.05 and *** P <0.001 versus basal; ^ P <0.05, ^^ P <0.01 and ^^^ P <0.001 versus relaxin/RXFP1; two-way ANOVA with Bonferroni post tests.

Article Snippet: Single-cell FRET data were analysed by two-way ANOVA with Bonferroni post tests and densitometric analyses, by one-way ANOVA with Newman–Keuls multiple comparison test (GraphPad Prism).

Techniques: Expressing, Activity Assay, Over Expression, Binding Assay, Control

Journal: The EMBO Journal

Article Title: Sub-picomolar relaxin signalling by a pre-assembled RXFP1, AKAP79, AC2, β-arrestin 2, PDE4D3 complex

doi: 10.1038/emboj.2010.168

Figure Lengend Snippet: Negative regulation of sub-picomolar relaxin signalling requires PKA, PDE4 and β-arrestin 2. Negative regulation was examined in HEK293 co-expressing RXFP1 or RXFP2, and pmEpac2, and stimulated with vehicle (0.001% TFA), 10 fM or 10 nM relaxin ( n =44–51). ( A ) The effect of PDE inhibition (IBMX, 100 μM, 30 min) on basal cAMP in cells expressing RXFP1 or RXFP2. ( B ) The 5 min area under the curve (AUC) from ( A ) for TFA, 10 fM and 10 nM relaxin. ( C ) The effect of IBMX (general inhibitor), Ro 20-1724 (PDE4 specific; 10 μM, 30 min) or cilostamide (PDE3 specific; 10 μM, 30 min) on the basal cAMP. ( D ) The 5 min AUC from ( C ) for TFA, 10 fM and 10 nM relaxin. ( E ) PKA was inhibited by H89 (10 μM, 30 min) or KT5720 (1 μM, 30 min) and the basal cAMP measured. ( F ) The 5 min AUC from ( E ) for TFA, 10 fM and 10 nM relaxin. ( G ) The effect of β-arrestin 1, β-arrestin 2 or scrambled siRNA (all 100 nM) on the basal cAMP response. ( H ) The 5 min AUC from ( G ) for TFA, 10 fM and 10 nM relaxin. ( I ) Western blots showing specific knockdown of β-arrestin 1 or β-arrestin 2 by targeted siRNA compared with controls. Blots were re-probed with tubulin to ensure equal protein loading . Data are expressed relative to the maximal cAMP response (FIP; 10 μM forskolin, 100 μM IBMX, 100 nM PGE 1 ), or as the 5 min AUC. Bars represent means, error bars s.e.m. * P <0.05, ** P <0.01 and *** P <0.001 versus basal; ^^ P <0.01 and ^^^ P <0.001 versus relaxin/pcDNA; two-way ANOVA with Bonferroni post tests.

Article Snippet: Single-cell FRET data were analysed by two-way ANOVA with Bonferroni post tests and densitometric analyses, by one-way ANOVA with Newman–Keuls multiple comparison test (GraphPad Prism).

Techniques: Expressing, Inhibition, Western Blot, Knockdown

Journal: The EMBO Journal

Article Title: Sub-picomolar relaxin signalling by a pre-assembled RXFP1, AKAP79, AC2, β-arrestin 2, PDE4D3 complex

doi: 10.1038/emboj.2010.168

Figure Lengend Snippet: Sub-picomolar relaxin signalling requires the RXFP1-transmembrane/C-terminus, negative regulation requires Ser 704 . The region of RXFP1 controlling sub-picomolar relaxin signalling was examined using mutant receptors in HEK293 cells expressing pmEpac2 or glEpac2 ( n =10–45). ( A ) Relaxin (10 aM–100 μM) or INSL3 (100 μM) stimulation of RXFP1/2 (RXFP1-ectodomain, RXFP2-transmembrane/C-terminus) co-expressed with pmEpac2. ( B ) Relaxin or INSL3 (10 aM–100 μM) stimulation of RXFP2/1 (RXFP2-ectodomain, RXFP1-transmembrane/C-terminus) co-expressed with pmEpac2. ( C ) The basal cAMP response in cells co-expressing RXFP1, tRXFP1-703 (truncated following helix 8; left) or RXFP1 S704A (potential phosphorylation site mutation; right) and glEpac2. ( D ) The 5 min area under the curve (AUC) from ( C ) for TFA, 10 fM and 10 nM relaxin. ( E ) The basal cAMP response in cells co-expressing RXFP1, tRXFP1-747 (truncation of final 10 residues; left) or RXFP1 S755A (potential phosphorylation site mutation; right) and glEpac2. ( F ) The 5 min AUC from ( E ) for TFA, 10 fM and 10 nM relaxin. Data are expressed relative to maximal cAMP (FIP; 10 μM forskolin, 100 μM IBMX, 100 nM PGE 1 ), or as the 5 min area AUC. Bars represent means, error bars s.e.m. * P <0.05, ** P <0.01 and *** P <0.001 relaxin versus basal; ^^ P <0.01 and ^^^ P <0.001 INSL3/mutant versus basal/RXFP1 respectively; two-way ANOVA, Bonferroni post tests.

Article Snippet: Single-cell FRET data were analysed by two-way ANOVA with Bonferroni post tests and densitometric analyses, by one-way ANOVA with Newman–Keuls multiple comparison test (GraphPad Prism).

Techniques: Mutagenesis, Expressing, Phospho-proteomics

Journal: The EMBO Journal

Article Title: Sub-picomolar relaxin signalling by a pre-assembled RXFP1, AKAP79, AC2, β-arrestin 2, PDE4D3 complex

doi: 10.1038/emboj.2010.168

Figure Lengend Snippet: The RXFP1 signalosome: AC2, AKAP79, β-arrestin 2, PKA and PDE4D3. Potential interactions between RXFP1 and mediators of the sub-picomolar relaxin response were assessed by GST pull down from HEK293 lysate using fragments of the RXFP1 C-terminus ( n =4), and confirmed in single-cell FRET studies. ( A ) Fragments of the RXFP1 C-terminus were tagged with GST. ( B ) Representative blots of GST pull-down assays probed with β-arrestin 2, PDE4D and PKAα-catalytic subunit antibodies . ( C ) Representative blots probed with AKAP79, AKAP149 and gravin antibodies . ( D ) Densitometry was calculated for β-arrestin 2, PDE4D3 and PKA. ( E ) Densitometry was calculated for AKAP79. Data are expressed as band density relative to pull down by GST alone. Bars represent means, error bars s.e.m. * P <0.05 and ** P <0.01 versus GST alone, two-way ANOVA with Bonferroni post tests. ( F ) Dominant negative PDE4D3 and PDE4D5 were overexpressed in HEK293 cells with RXFP1 and pmEpac2 for single-cell FRET studies. The 5 min area under the curve (AUC) was calculated for TFA, 10 fM and 10 nM relaxin ( n =16–43). Western blotting shows expression of the two isoforms with equal loading confirmed using a tubulin antibody . ( G ) The effects of AKAP79 shRNA or pSilencer control were assessed in HEK293 cells co-expressing RXFP1 and pmEpac2 using single-cell FRET studies. The 5 min AUC was calculated for TFA, 10 fM and 10 nM relaxin ( n =20–51). Western blotting shows decreased AKAP79 protein following shRNA transfection with equal protein loading confirmed using a tubulin antibody . Bars represent means, error bars s.e.m. * P <0.05 and *** P <0.001 versus own basal; ^ P <0.05 and ^^ P <0.01 versus control; two-way ANOVA with Bonferroni post tests.

Article Snippet: Single-cell FRET data were analysed by two-way ANOVA with Bonferroni post tests and densitometric analyses, by one-way ANOVA with Newman–Keuls multiple comparison test (GraphPad Prism).

Techniques: Dominant Negative Mutation, Western Blot, Expressing, shRNA, Control, Transfection