Journal: PloS one

Article Title: β2 adrenergic receptor fluorescent protein fusions traffic to the plasma membrane and retain functionality.

doi: 10.1371/journal.pone.0074941

Figure Lengend Snippet: Figure 1. OP 6 cells transiently expressing gap::XFPs and β2AR::XFPs. Single cells expressing each gap tagged fluorescent protein localizes to the plasma membrane (A-G). gap::XFPs also target to the filopodia (arrowheads, Ai-Gi magnified images). Single cells expressing each β2AR fusion (β2AR::XFP) (H-N) exhibit diffuse protein expression at the plasma membrane defined by localization at the filopodia (arrowheads, Hi-Ni magnified images). The ability of β2AR to traffic to the plasma membrane is unaffected by fusion to the seven tested fluorescent proteins.

Article Snippet: For detection of wild type β2AR, antibody against β2AR (goat; Santa Cruz Cat# sc-570) was used at a 1:100 dilution followed by a 1:500 dilution of goat anti-rabbit Alexa Fluor 488 secondary antibody.

Techniques: Expressing, Clinical Proteomics, Membrane

Journal: PloS one

Article Title: β2 adrenergic receptor fluorescent protein fusions traffic to the plasma membrane and retain functionality.

doi: 10.1371/journal.pone.0074941

Figure Lengend Snippet: Figure 2. Filopodia count in OP 6 cells transiently expressing untagged fluorescent proteins, gap::XFPs, or β2AR::XFPs. Filopodia on N=10 cells were counted for cells expressing each untagged fluorescent protein, tagged with gap, or fused to the β2AR. gap::XFPs featured 78-141 filopodia per 10 cells, and β2AR::XFPs featured 97-167 filopodia per 10 cells. Cells expressing the untagged fluorescent proteins featured between 7-16 filopodia. Expression in filopodia of gap::XFPs and β2AR::XFPs was significantly different from the untagged XFPs, Fisher’s exact test P<0.0001.

Article Snippet: For detection of wild type β2AR, antibody against β2AR (goat; Santa Cruz Cat# sc-570) was used at a 1:100 dilution followed by a 1:500 dilution of goat anti-rabbit Alexa Fluor 488 secondary antibody.

Techniques: Expressing

Journal: PloS one

Article Title: β2 adrenergic receptor fluorescent protein fusions traffic to the plasma membrane and retain functionality.

doi: 10.1371/journal.pone.0074941

Figure Lengend Snippet: Figure 3. Colocalization of β2AR::XFP and transferrin after isoprenaline exposure. Single OP 6 cells expressing each β2AR::XFP construct were incubated with 20µg/ml of Alexa Fluor transferrin 647 (A-E) or Alexa Fluor transferrin 488 (F, G) for 30 minutes and then exposed to 10µM isoprenaline for 20 minutes. β2AR::XFP is diffusely expressed at the plasma membrane, and transferrin is localized in the endosomes (A-G, 0) before isoprenaline exposure. After 20 minutes, β2AR::XFP is internalized and becomes punctate, colocalizing with transferrin (arrowheads). ICQ values for all fusions reflect a significant increase (P<0.0001) in dependency after exposure to isoprenaline. ICQ values for 0 minute and 20 minute isoprenaline stimulation: (A) 0.086, 0.186 (B) 0.046, 0.187 (C) 0.116, 0.227 (D) -0.006, 0.212 (E) 0.132, 0.319 (F) 0.013, 0.254 (G) 0.047, 0.133.

Article Snippet: For detection of wild type β2AR, antibody against β2AR (goat; Santa Cruz Cat# sc-570) was used at a 1:100 dilution followed by a 1:500 dilution of goat anti-rabbit Alexa Fluor 488 secondary antibody.

Techniques: Expressing, Construct, Incubation, Clinical Proteomics, Membrane

Journal: PloS one

Article Title: β2 adrenergic receptor fluorescent protein fusions traffic to the plasma membrane and retain functionality.

doi: 10.1371/journal.pone.0074941

Figure Lengend Snippet: Figure 4. Colocalization of β2AR::XFP and β-Arrestin2 after isoprenaline exposure. Single OP 6 cells expressing β2AR::XFP and myc-tagged β-Arrestin2 were fixed after 24 hours and incubated with rabbit polyclonal antibody for Myc in the absence of isoprenaline, or after 20 minutes of exposure to 10µM isoprenaline. Anti-rabbit Alexa Fluor 546 was used for A- E, and anti-rabbit Alexa Fluor 488 was used for F, G. In the absence of isoprenaline (0 minutes), β2AR::XFP exhibits diffuse expression at the plasma membrane (A-G). After 20 minutes of exposure to 10µM isoprenaline, β2AR::XFP becomes punctate and colocalizes with β-Arrestin2 (arrowheads). ICQ values for all fusions reflect a significant increase (P<0.0001) in dependency after isoprenaline exposure. ICQ values for 0 minute and 20 minute isoprenaline stimulation: (A) 0.045, 0.107 (B) 0.098, 0.375 (C) 0.01, 0.17 (D) 0.136, 0.325 (E) 0.199, 0.381 (F) 0.014, 0.215 (G) -0.014, 0.139.

Article Snippet: For detection of wild type β2AR, antibody against β2AR (goat; Santa Cruz Cat# sc-570) was used at a 1:100 dilution followed by a 1:500 dilution of goat anti-rabbit Alexa Fluor 488 secondary antibody.

Techniques: Expressing, Incubation, Clinical Proteomics, Membrane

Journal: PloS one

Article Title: β2 adrenergic receptor fluorescent protein fusions traffic to the plasma membrane and retain functionality.

doi: 10.1371/journal.pone.0074941

Figure Lengend Snippet: Figure 5. Detailed time course analysis of β2AR::Teal and β2AR::Venus. ICQ values were used to measure colocalization events. (A) Single OP 6 cells (n=2) expressing the β2AR::Teal or β2AR::Venus construct were incubated with 20µg/ml of Alexa Fluor transferrin 647 for 30 minutes and then exposed to 10µM isoprenaline and imaged live every 4 minutes until 20 minutes. (B) Single OP 6 cells (n=3,4) expressing the β2AR::Teal or β2AR::Venus construct and myc-tagged β- Arrestin2 were fixed after 24 hours and incubated with rabbit polyclonal antibody for Myc after exposure to 10µM isoprenaline in 4 minute intervals between 0 and 20 minutes and were visualized with Anti-rabbit Alexa Fluor 546 antibody. Fusions of Teal and Venus to the β2AR follow the same time course for internalization into the early endosomes (A) and the same time course regulation by β-Arrestin2 (B). Error bars represent standard error.

Article Snippet: For detection of wild type β2AR, antibody against β2AR (goat; Santa Cruz Cat# sc-570) was used at a 1:100 dilution followed by a 1:500 dilution of goat anti-rabbit Alexa Fluor 488 secondary antibody.

Techniques: Expressing, Construct, Incubation

Journal: PloS one

Article Title: β2 adrenergic receptor fluorescent protein fusions traffic to the plasma membrane and retain functionality.

doi: 10.1371/journal.pone.0074941

Figure Lengend Snippet: Figure 6. Dose response curves for β2AR::XFPs. Cells expressing each β2AR::XFP and human Gα15 were exposed to concentrations of isoprenaline and analyzed using the FLIPR assay. Normalized curves (A) show an EC50 between 2.6-5.2 x 10-9

Article Snippet: For detection of wild type β2AR, antibody against β2AR (goat; Santa Cruz Cat# sc-570) was used at a 1:100 dilution followed by a 1:500 dilution of goat anti-rabbit Alexa Fluor 488 secondary antibody.

Techniques: Expressing

Journal: PloS one

Article Title: β2 adrenergic receptor fluorescent protein fusions traffic to the plasma membrane and retain functionality.

doi: 10.1371/journal.pone.0074941

Figure Lengend Snippet: Figure 7. Visualization of OP 6 cells transiently expressing individual β2AR::XFPs and mixed. Mixed OP 6 cells expressing [β2AR::Cerulean, β2AR::Venus, β2AR::mCherry] in (A), [β2AR::Teal, β2AR::Venus, β2AR::mCherry] in (B) and [β2AR::GFP, β2AR::mCherry, β2AR::AFP] in (C). Each β2AR::XFP fusion was solely identified in its respective channel (Ai, Cerulean, Bi, Venus, Ci, mCherry), (Aii, Teal, Bii, Venus, Cii, mCherry), (Aiii, GFP, Biii, mCherry, Ciii, AFP).

Article Snippet: For detection of wild type β2AR, antibody against β2AR (goat; Santa Cruz Cat# sc-570) was used at a 1:100 dilution followed by a 1:500 dilution of goat anti-rabbit Alexa Fluor 488 secondary antibody.

Techniques: Expressing

Journal: Heliyon

Article Title: Mangiferin inhibits chronic stress-induced tumor growth in colorectal liver metastases via WAVE2 signaling pathway

doi: 10.1016/j.heliyon.2023.e13753

Figure Lengend Snippet: MGF inhibits activation of HSCs via the WAVE2 signaling pathway. (A) Transduced HSCs treated with TGF-1 (2.5 ng/mL) and IF for SMA show that MGF consistently suppresses TGF-1-induced myofibroblast differentiation. (B) Following 24 h of serum starvation and treatment with TGF-1, control and MGF-treated HSCs are shown. Western blot analysis for detection of HSC activation markers, α-SMA and p -SMAD2, and activator β2-AR and TGF-β1. Data are presented as the mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The stock solution was used to prepare different concentrations in the test media. including β2-AR polyclonal antibody (Cat. #8513, CST), WAVE2 polyclonal antibody (Cat. # YT4898, ImmunoWay), VEGF polyclonal antibody (Cat. # sc-7269, Santa Cruz Biotechnology), α-smooth muscle actin (α-SMA) (Abcam, Cat. # ab5694), a polyclonal antibody against p -SMAD2 (Cat. #3104, CST) at a 1:1000 and GAPDH mouse monoclonal antibody (Cat. # ab8245, Abcam) at 1:5000 dilution.

Techniques: Activation Assay, Control, Western Blot