cell culture camp assay  (GraphPad Software Inc)

Journal: Science signaling

Article Title: A Network of G Protein α i Signaling Partners is Revealed by Proximity Labeling Proteomics and Includes PDZ-RhoGEF

doi: 10.1126/scisignal.abi9869

Figure Lengend Snippet: (A) Left: Schematic of BioID2 fusion constructs. BioID2 was inserted between residues Ala121-Glu122 in the αb-αc loop (the first loop of the helical domain) of human Gαi1, flanked by SG-linkers. Palmitoylation and myristylation sites on the Gαi1 subunit and the farnesylation sites on CaaX moiety are labeled as lipid modifications. Right: Schematic of principle and experimental workflow of proximity-based labeling using BioID2. HT1080 cells were used for mass spectrometry experiments and A293 cells were used for pull-down western blot. Cells were transfected with the indicated constructs and labeled for 24 hr in the presence of biotin. Gαi1 fused BioID2 biotinylates proteins in proximity (< 20 nm) in an unbiased manner to identify candidate interacting proteins of Gαi1. (B) A293 cells were transfected with indicated constructs and labeled with biotin for 24 hr. Top: Biotinylated proteins in whole-cell lysates were detected on a streptavidin Western blot. The two bands at 130 and ~90 kDa correspond to endogenously biotinylated proteins in control lanes. Middle: Cell lysates were immunoblotted with Gαi1/2 antisera to detect BioID2-Gαi1 and BioID2-Gαi1-QL and with Myc antibody to detect BioID2-CaaX. Bottom: Ponceau S-stained blot showing total protein loading. Western blots are representative of three independent experiments that yielded similar results. (C) Schematic of sample processing and mass spectrometry analysis. Samples pulled down using streptavidin beads were digested with trypsin and labelled with a TMT tag. Biological triplicate samples of lysates of cells expressing BioID2-Gαi1, BioID2-Gαi1-QL and BioID2-CaaX were pooled and resolved by LC-MS and the data was analyzed using proteome discover.

Article Snippet: Glosensor cAMP Reporter Assay A293 cells (4 × 10 4 cells/well) were plated per well in a 96-well plate (655983, Greiner).

Techniques: Construct, Labeling, Mass Spectrometry, Western Blot, Transfection, Staining, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: Science signaling

Article Title: A Network of G Protein α i Signaling Partners is Revealed by Proximity Labeling Proteomics and Includes PDZ-RhoGEF

doi: 10.1126/scisignal.abi9869

Figure Lengend Snippet: (A) Normalized abundance of PRG was quantified by MS in cells expressing BioID2-Gαi1, BioID2-Gαi1-QL, or BioID2-CaaX. The data represent the mean ± SD of three independent experiments. (****P<0.0001, one-way ANOVA with Tukey’s multiple comparisons test). (B) HT1080 cells were transfected with the indicated constructs and whole cell lysates were resolved by Western blot. (C) A293 cells were transfected with PRG and BioID2-Gαi1, BioID2-Gαi1-QL, or BioID2-CaaX and labeled with biotin for 24 hr. Cell lysates were subjected to streptavidin pulldowns. Left: Representative Western blots of PRG in streptavidin pull-downs from cells expressing BioID2-Gαi1, BioID2-Gαi1-QL, or BioID2-CaaX. Right: Quantitation (shown as mean ± SD) of three independent experiments normalized to total PRG. ***P < 0.001, ****<0.0001, one-way ANOVA with Tukey’s multiple comparisons test). (D) PLAs were performed in cells transfected with GFP-PRG and APEX2-FLAG-tagged Gαi1-WT, Gαi1-QL, or CaaX. Left: Representative images from three randomly selected fields show GFP-PRG (green), PLA reaction (red), merge (orange) and DAPI (blue). Scale bar, 10μm. Right: The intensity of the PLA signal (y-axis) was plotted against GFP-PRG expression (x-axis). For each experiment, ~100 cells per condition were analyzed, and the data are shown from one of three independent experiments that yielded similar results (See fig. S5).

Article Snippet: Glosensor cAMP Reporter Assay A293 cells (4 × 10 4 cells/well) were plated per well in a 96-well plate (655983, Greiner).

Techniques: Expressing, Transfection, Construct, Western Blot, Labeling, Quantitation Assay

Journal: Science signaling

Article Title: A Network of G Protein α i Signaling Partners is Revealed by Proximity Labeling Proteomics and Includes PDZ-RhoGEF

doi: 10.1126/scisignal.abi9869

Figure Lengend Snippet: (A) A293 cells were transfected with cDNAs encoding SRE-Luciferase, PRG, and either Gαi1 or Gαi1-QL for 20 hr. Left and middle: Luminescence was measured in serum-starved cells 10 min after the addition of One-Glo™ reagent. The data represent the mean ± SD of three independent experiments. Right: Representative Western blots showing relative expression of various cDNA constructs in A293 cells. Data are representative of three independent experiments that yielded similar results. (B) A293 cells were transfected with the indicated cDNA constructs and cell lysates were incubated with GST-Rhotekin beads. Left: Representative Western blots showing bound RhoA-GTP and relative expression of transfected constructs. Right: Quantification (shown as mean ± SD) of three independent experiments, normalized to total RhoA. (**P < 0.01, ****<0.0001, Panel A left and B: one-way ANOVA with Tukey’s multiple comparisons test, Panel A middle: two-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Glosensor cAMP Reporter Assay A293 cells (4 × 10 4 cells/well) were plated per well in a 96-well plate (655983, Greiner).

Techniques: Transfection, Luciferase, Western Blot, Expressing, Construct, Incubation

Journal: Science signaling

Article Title: A Network of G Protein α i Signaling Partners is Revealed by Proximity Labeling Proteomics and Includes PDZ-RhoGEF

doi: 10.1126/scisignal.abi9869

Figure Lengend Snippet: (A) A293 cells were transfected with cDNAs encoding SRE-Luciferase, PRG, and either Gαi1-QL, Gαi2-QL, or Gαi3-QL. Luminescence was measured in serum-starved cells 10 min after the addition of One-Glo™ reagent. The data represent the mean ± SD of three independent experiments. (B) Western blot showing relative expression of Gαi1 and Gαi2 constructs in A293 cells. Data are representative of three independent experiments that yielded similar results. (C) Cells were transfected with cAMP Glosensor™ and WT and QL versions of Gαi1, Gαi2, or Gαi3 for 24 hr. Luminescence was measured for 30 min (x-axis) after Forskolin (Fsk) stimulation and represented as % stimulation (y-axis) relative to the maximum signal in the respective WT group with 1 μM Fsk treatment. Data shown as mean ±SD are representative of three independent experiments (See fig. S5A for replicate experiments). (D) A293 cells were transfected with PRG and BioID2-Gαi1-QL, BioID2-Gαi3-QL, or BioID2-Gαi2-QL and labeled with biotin for 24 hr. Cell lysates were subjected to streptavidin pulldowns. Left: Representative Western blots of PRG in streptavidin pull-downs as well as of expression of BioID2-Gαi1, Gαi1-QL and BioID2-CaaX. Right: Quantitation shown as mean ± SD of three independent experiments normalized to total PRG. (***P <0.001, ****<0.0001, one-way ANOVA with Tukey’s multiple comparisons test).

Article Snippet: Glosensor cAMP Reporter Assay A293 cells (4 × 10 4 cells/well) were plated per well in a 96-well plate (655983, Greiner).

Techniques: Transfection, Luciferase, Western Blot, Expressing, Construct, Labeling, Quantitation Assay

Journal: Science signaling

Article Title: A Network of G Protein α i Signaling Partners is Revealed by Proximity Labeling Proteomics and Includes PDZ-RhoGEF

doi: 10.1126/scisignal.abi9869

Figure Lengend Snippet: (A) A293 cells stably expressing FPR1 (A293-FPR1) were transfected with cDNAs encoding SRE-Luc, PRG, and Gαi1-WT or Gαi2-WT and incubated for 12 hr in serum free media containing fMLF (0.01, 0.1, 1 and 10 μM) or DMSO. Data from three independent experiments were plotted as mean ± SD. (B) A293-FPR1 cells were transfected and incubated for 12 hours in serum-free media containing PTX (100 ng/mL) and fMLF (10 μM). Luminescence was measured 10 min after the addition of the One-Glo™ reagent. The data combined data (mean ± SD) of three independent experiments. (C) A293-FPR1 cells were transfected with PRG, Gαi1-WT, and YFP for 36 hr. 24 hr after transfection, cells were treated with PTX (100 ng/mL) for 12 hr. Cells were stimulated with 100 nM fMLF and live cell video microscopy was performed for 40 min. Left: Representative images of A293-FPR1 cells expressing PRG + Gαi1-WT and treated with fMLF or DMSO are shown. Scale bar, 100 μm. Right: Quantitative analysis of the percentage of cells with dynamic protrusions shown as mean ± SD from three independent experiments. For each experiment, >500 cells per condition were analyzed in a blinded manner. (D) Human neutrophils were pretreated or not with PTX (500 ng/mL) for 2 hr, allowed to adhere to the fibronectin-coated surface for 15 min, and stimulated with 10 nM fMLF for 5 min. The cells were stained with a P-MLC antibody and DAPI and imaged with confocal microscopy. Left: Representative images from three randomly selected fields of view are shown. Right: Total number of cells and cells with asymmetric P-MLC localization were counted in a field and the % cells with polarized P-MLC localization (mean ± SD) from three independent experiments were plotted. (****p < 0.0001, Panel A: Two-way ANOVA with Tukey’s multiple comparisons test, Panel B, C, D: One-way ANOVA with Tukey’s multiple comparison test). Scale bar, 10 μm.

Article Snippet: Glosensor cAMP Reporter Assay A293 cells (4 × 10 4 cells/well) were plated per well in a 96-well plate (655983, Greiner).

Techniques: Stable Transfection, Expressing, Transfection, Incubation, Microscopy, Staining, Confocal Microscopy