primary antibodies for β2-ar  (Proteintech)

Journal: International Journal of Molecular Sciences

Article Title: Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

doi: 10.3390/ijms22137232

Figure Lengend Snippet: Pre-administration of the β2-AR agonist (salbutamol) protects against Meth toxicity. This effect is occluded by the β2-AR antagonist (butoxamine). The selective β2-AR agonist salbutamol (“Sal”, 5 μM) fully protects against the toxicity induced by Meth (50 μM). This protective effect is occluded in the presence of the selective β2-AR antagonist butoxamine (“Butox”, 10 μM). Butoxamine also fully prevents NE (5 nM)-induced protection against Meth toxicity, while it does not significantly modify Meth toxicity or spontaneous toxicity ongoing in control cells when administered alone. The effects on cell mortality of the β2-AR antagonist butoxamine (“Butox”) were assessed on naïve PC12 cells and in PC12 cells exposed to Meth (50 μM) and/or NE (5 nM). NE was used, either alone or in the group NE + Meth and “NE+Butox + Meth” starting 30 min before Meth administration; butoxamine was administered 15 min before NE in the group “NE+Butox + Meth” or 45 min before Meth in the group “Butox + Meth”. PC12 cells were stained at 72 h after Meth. ( A ) Graph reporting TB staining; ( B ) graph reporting H&E staining; ( C ) graph reporting FJB staining. Data are given as the mean + SEM of nine independent counts for TB; six independent counts for H&E and FJB. Inferential statistics was carried out with ANOVA with Scheffè’s post hoc analysis. (DF = 15) * p < 0.05 compared with controls. ** p ≤ 0.05 compared with Meth.

Article Snippet: Subsequently, the membrane was incubated overnight at 4 °C on the plate shaker with the following primary antibodies: rabbit anti-LC3-I/LC3-II (1:1000; Cell Signaling), rabbit anti-p62 (1:1000; Sigma), and rabbit anti-β2-AR primary antibody (1:2000, Bioss Antibodies Inc., Woburn, MA, USA).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

doi: 10.3390/ijms22137232

Figure Lengend Snippet: The β2-AR agonist (salbutamol) protects against Meth toxicity, while the β2-AR antagonist (butoxamine) impedes such a protection (H&E staining). Representative pictures show that salbutamol (5 μM) protects against Meth-induced toxicity. This β2-AR agonist is no longer protecting when the selective β2-AR antagonist (butoxamine) is administered. Butoxamine alone (10 μM) does not alter cell viability. Arrows indicate pale eosinophilic cells. Scale bar = 8 μM.

Article Snippet: Subsequently, the membrane was incubated overnight at 4 °C on the plate shaker with the following primary antibodies: rabbit anti-LC3-I/LC3-II (1:1000; Cell Signaling), rabbit anti-p62 (1:1000; Sigma), and rabbit anti-β2-AR primary antibody (1:2000, Bioss Antibodies Inc., Woburn, MA, USA).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

doi: 10.3390/ijms22137232

Figure Lengend Snippet: The β2-AR agonist (salbutamol) protects against Meth toxicity, while the β2-AR antagonist (butoxamine) impedes such a protection (FJB staining). Representative pictures show that salbutamol (5 μM) protects against Meth-induced toxicity. In fact, intensely fluorescent Meth-treated PC12 cells turn out into dark cells when salbutamol is pre-administered. This β2-AR agonist is no longer protecting when the selective β2-AR antagonist (butoxamine) is administered. In fact, cells administered salbutamol + butoxamine + Meth revert into intensely yellow fluorescence. Arrows indicate FJB intensely positive cells. Scale bar = 20 μM.

Article Snippet: Subsequently, the membrane was incubated overnight at 4 °C on the plate shaker with the following primary antibodies: rabbit anti-LC3-I/LC3-II (1:1000; Cell Signaling), rabbit anti-p62 (1:1000; Sigma), and rabbit anti-β2-AR primary antibody (1:2000, Bioss Antibodies Inc., Woburn, MA, USA).

Techniques: Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

doi: 10.3390/ijms22137232

Figure Lengend Snippet: Protection of the NET blocker DMI on Meth toxicity through β2-AR is reverted by butoxamine. When the blocker of the NE transporter (NET) desmethylimipramine (DMI) was administered at the dose of 100 nM to PC12 cells, no effect could be detected. However, DMI 100 nM markedly protects against toxicity induced by Meth 50 μM to an amount that was comparable to NE 5 nM. Such an effect was lost when the β2-AR antagonist butoxamine 10 μM was pre-administered. This experiment was carried out to assess the contribution of extracellular vs. intracellular NE to protect against Meth toxicity. One might argue that an NET blocker reduces the amount of Meth that enters the cells. However, the passive diffusion of Meth and the entry through the dopamine transporter (DAT) are expected to overcome such a caveat. Most significantly, when the protection was due to a block in the entry of Meth, this could not be reversed by the β2-AR antagonist butoxamine. These data indicate that NE needs to act externally to the cell by activating β2-AR in order to protect against Meth toxicity. When co-administered with Meth, NE (5 nm) was applied 30 min before Meth; DMI (100 nM) or butoxamine (10 μM) were administered 45 min before Meth in the combined groups. ( A ) Graph reporting TB staining; ( B ) graph reporting H&E staining; ( C ) graph reporting FJB staining. Data are given as the mean+SEM of nine independent counts for TB; six independent counts for H&E and FJB. Inferential statistics was carried out with ANOVA with Scheffè’s post hoc analysis. (DF = 15). * p < 0.05 compared with controls. ** p ≤ 0.05 compared with Meth.

Article Snippet: Subsequently, the membrane was incubated overnight at 4 °C on the plate shaker with the following primary antibodies: rabbit anti-LC3-I/LC3-II (1:1000; Cell Signaling), rabbit anti-p62 (1:1000; Sigma), and rabbit anti-β2-AR primary antibody (1:2000, Bioss Antibodies Inc., Woburn, MA, USA).

Techniques: Diffusion-based Assay, Blocking Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

doi: 10.3390/ijms22137232

Figure Lengend Snippet: The NET blocker DMI protects against Meth toxicity through β2-AR (H&E). Representative pictures show that the NET inhibitor DMI (100 nM) protects against Meth (50 μM)-induced toxicity. This NET blocker is no longer protecting when the selective β2-AR antagonist (butoxamine) is administered. Butoxamine (10 μM) does not alter cell viability. Arrows indicate pale eosinophilic cells. Scale bar = 8 μM.

Article Snippet: Subsequently, the membrane was incubated overnight at 4 °C on the plate shaker with the following primary antibodies: rabbit anti-LC3-I/LC3-II (1:1000; Cell Signaling), rabbit anti-p62 (1:1000; Sigma), and rabbit anti-β2-AR primary antibody (1:2000, Bioss Antibodies Inc., Woburn, MA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

doi: 10.3390/ijms22137232

Figure Lengend Snippet: The NET blocker DMI protects against Meth toxicity through β2-AR (FJB). DMI (100 nM) administration obscures intense histofluorescence by FJB staining induced during Meth toxicity. Histofluorescence is brought back in intensity by the β2-AR antagonist butoxamine (10 μM), which re-instates Meth (50 μM)-induced toxicity. Arrows indicate FJB intensely positive cells. Scale bar = 20 μM.

Article Snippet: Subsequently, the membrane was incubated overnight at 4 °C on the plate shaker with the following primary antibodies: rabbit anti-LC3-I/LC3-II (1:1000; Cell Signaling), rabbit anti-p62 (1:1000; Sigma), and rabbit anti-β2-AR primary antibody (1:2000, Bioss Antibodies Inc., Woburn, MA, USA).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

doi: 10.3390/ijms22137232

Figure Lengend Snippet: Transfection with β2-AR siRNA suppresses the protective effects of NE against Meth. Cell viability in naive and β2-AR siRNA- and scRNA-transfected cells, as evaluated by ( A ) TB, ( B ) H&E, and ( C ) WST-1 assay. ( D ) Dissipation of LC3 particles from vacuoles to the cytosol in β2-AR silenced cells. (DF = 11) * p < 0.05 compared with control; ** p < 0.05 compared with Meth.

Article Snippet: Subsequently, the membrane was incubated overnight at 4 °C on the plate shaker with the following primary antibodies: rabbit anti-LC3-I/LC3-II (1:1000; Cell Signaling), rabbit anti-p62 (1:1000; Sigma), and rabbit anti-β2-AR primary antibody (1:2000, Bioss Antibodies Inc., Woburn, MA, USA).

Techniques: Transfection, WST-1 Assay

Journal: International Journal of Molecular Sciences

Article Title: Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

doi: 10.3390/ijms22137232

Figure Lengend Snippet: Meth-induced FJB fluorescence is not reduced by NE after transfection with β2-AR siRNA. ( A ) Representative pictures and FJB fluorescent cells showing the effects of Meth and NE in naïve and β2-AR siRNA- and scRNA-transfected cells. ( B ) The graph reports the number of FJB fluorescent cells in the different experimental groups. (DF = 11) * p < 0.05 compared with control; ** p < 0.05 compared with Meth. Scale bar = 13.3 μM.

Article Snippet: Subsequently, the membrane was incubated overnight at 4 °C on the plate shaker with the following primary antibodies: rabbit anti-LC3-I/LC3-II (1:1000; Cell Signaling), rabbit anti-p62 (1:1000; Sigma), and rabbit anti-β2-AR primary antibody (1:2000, Bioss Antibodies Inc., Woburn, MA, USA).

Techniques: Fluorescence, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

doi: 10.3390/ijms22137232

Figure Lengend Snippet: Opposite effects of NE and the β2-AR antagonist butoxamine on the LC3-II and p62 accumulation induced by Meth. Western blot for the autophagy markers LC3-I and LC3-II ( A ) and p62 ( B ) are reported along with the related optical densities. (DF = 7). * p < 0.05 compared with controls; ** p < 0.05 compared with Meth. Ctrl = control.

Article Snippet: Subsequently, the membrane was incubated overnight at 4 °C on the plate shaker with the following primary antibodies: rabbit anti-LC3-I/LC3-II (1:1000; Cell Signaling), rabbit anti-p62 (1:1000; Sigma), and rabbit anti-β2-AR primary antibody (1:2000, Bioss Antibodies Inc., Woburn, MA, USA).

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

doi: 10.3390/ijms22137232

Figure Lengend Snippet: Effects of NE, Meth, β2-AR ligands, and DMI on autophagy vacuoles and LC3 compartmentalization. The graphs show ( A ) the amount of authentic autophagy vacuoles (defined as double/multiple membrane vacuoles containing LC3 molecules); ( B ) the amount of whole cell LC3 immunogold particles; ( C ) LC3 positive vacuoles; ( D ) the ratio between LC3 within vacuoles and whole cell LC3. Measurements are obtained by ultrastructural morphometry where the quantitative measurement of vacuoles and stoichiometrically identified LC3 particles by immunogold are counted. The graphs show that Meth increases the amount of autophagy-like vacuoles and autophagy vacuoles along with whole cells LC3 particles compared with control. Nonetheless, the ratio of LC3 within vacuoles and LC3 in the whole cell is strongly diminished. This indicates a de-polarization of LC3 from vacuoles to the cytosol. Such an effect is reversed by NE, which by itself does not increase polarization compared with controls. Salbutamol replicates the data obtained with NE, as it does DMI, while butoxamine occludes these effects. Numbers are given as the mean+SEM of autophagy-like vacuoles/autophagy vacuoles/cytosolic LC3 particles/vacuolar LC3 particles per cell, which were counted in 50 cells per group. Inferential statistics was carried out with ANOVA with Scheffè’s post hoc analysis. (DF = 16). * p < 0.05 compared with controls. ** p ≤ 0.05 compared with Meth.

Article Snippet: Subsequently, the membrane was incubated overnight at 4 °C on the plate shaker with the following primary antibodies: rabbit anti-LC3-I/LC3-II (1:1000; Cell Signaling), rabbit anti-p62 (1:1000; Sigma), and rabbit anti-β2-AR primary antibody (1:2000, Bioss Antibodies Inc., Woburn, MA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

doi: 10.3390/ijms22137232

Figure Lengend Snippet: NE and the β2-AR agonist salbutamol do not counteract the inhibition of the autophagy flux produced by bafilomycin. Western blot for LC3-I and LC3-II ( A ) and related optical density ( B ) are reported. (DF = 5). * p < 0.05 compared with controls.

Article Snippet: Subsequently, the membrane was incubated overnight at 4 °C on the plate shaker with the following primary antibodies: rabbit anti-LC3-I/LC3-II (1:1000; Cell Signaling), rabbit anti-p62 (1:1000; Sigma), and rabbit anti-β2-AR primary antibody (1:2000, Bioss Antibodies Inc., Woburn, MA, USA).

Techniques: Inhibition, Produced, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

doi: 10.3390/ijms22137232

Figure Lengend Snippet: The cartoon summarizes the protective effects of NE against Meth-induced toxicity. ( A ) In detail, Meth moves out LC3 molecules from autophagy vacuoles toward the cytosol, thus de-potentiating the autophagy machinery. ( B ) NE, by acting on β2-AR, counteracts Meth-induced toxicity while re-polarizing LC3 toward autophagy vacuoles.

Article Snippet: Subsequently, the membrane was incubated overnight at 4 °C on the plate shaker with the following primary antibodies: rabbit anti-LC3-I/LC3-II (1:1000; Cell Signaling), rabbit anti-p62 (1:1000; Sigma), and rabbit anti-β2-AR primary antibody (1:2000, Bioss Antibodies Inc., Woburn, MA, USA).

Techniques:

Journal: bioRxiv

Article Title: Insulin inhibits protein phosphatase 2A to impair β-adrenergic receptor function

doi: 10.1101/2020.09.17.302448

Figure Lengend Snippet: (A) Immunoblotting performed on cell lysates with anti-phospho-β2AR antibody to assess phosphorylation of β2AR following saline (Sal), isoproterenol (ISO, 10uM ) or insulin (INS, 100nM ) stimulation for 10 minutes in FLAG-β2AR HEK 293 cells. Cumulative densitometry is shown in the bar graph. (n=5) *p< 0.0001 versus control (Sal). Actin was used as loading control for all experiments. (B) FLAG-β2AR phosphorylation was visualized by confocal microscopy using anti-phospho-β2AR antibody (green) following ISO or INS stimulation for 10 minutes. Only ISO treated cells were pre-treated with internalization blockers (sucrose and β-cyclodextrin) to assess membrane decoration of stained receptors as positive control while, INS treated cells were not pre-treated with endocytosis blockers. Nucleus was visualized by DAPI (blue) staining. Scale bar: 10 μm. (C) Immunoblotting was performed on plasma membrane fraction to assess β2AR phosphorylation in FLAG-β2AR HEK 293 cells following INS stimulation for 10 minutes. Cumulative data is shown in the bar graph, (n=4) *p< 0.001 versus control. (D) Role of PI3K in INS-mediated β2AR phosphorylation was determined by pre-treatment with PI3K-specific inhibitors LY294002 (1uM) or Wortmannin (100nM). β2AR phosphorylation was assessed by immunoblotting with anti-phospho-β2AR antibody following INS stimulation for 10 minutes in HEK-FLAG-β2AR cells. (E) To specifically test the role of PI3Kγ in INS-mediated β2AR phosphorylation, HEK 293 cells with stable knock-down of PI3Kγ (PI3Kγ KD) were assessed for β2AR phosphorylation by immunoblotting with anti-phospho-β2AR antibody following the INS stimulation for 10 minutes. Cumulative data is shown in the bar graph below, (n=6), *p< 0.001 versus control (Sal). (F) Immunoblots to assess phospho-β2AR following INS stimulation for 10 minutes in mouse embryonic fibroblasts isolated from wild-type mice (WT MEF) and PI3Kγ KO mice (KO MEF). Cumulative densitometry data is shown in the bar graph, (n=5), *p< 0.001 versus control (Sal).

Article Snippet: The membranes were blocked with 5% BSA and immunoblotted using primary anti-phospho-β2AR antibody (1:1000) (Santa Cruz Biotechnology), anti-PI3Kγ (1:500) (Santa Cruz Biotechnology), anti-GRK2 (1:1000) (Santa Cruz Biotechnology), anti-I2PP2A (1:700) (Santa Cruz Biotechnology or) or anti-PP2Ac (1:3000) (Santa Cruz Biotechnology).

Techniques: Western Blot, Phospho-proteomics, Saline, Control, Confocal Microscopy, Membrane, Staining, Positive Control, Clinical Proteomics, Knockdown, Isolation

Journal: bioRxiv

Article Title: Insulin inhibits protein phosphatase 2A to impair β-adrenergic receptor function

doi: 10.1101/2020.09.17.302448

Figure Lengend Snippet: - β2AR phosphorylation in H9c2 cardiac myoblasts following INS stimulation: H9c2 cardiac myoblasts were serum starved for 4 hours followed by stimulation with classical β2AR agonist isoproterenol (ISO, 10 μM) or insulin (INS, 100 nM). H9c2 cell lysates were immunoblotted with anti-phospho-β2AR antibody to assess β2AR phosphorylation. Significant β2AR phosphorylation of endogenous β2ARs is observed following ISO or INS. Cumulative data for INS-mediated phosphorylation is presented in the bar graphs. (n = 3). *p < 0.001 vs. saline control (Sal). β-actin was used as loading control. - PI3Kγ expression level in PI3Kγ stable knockdown and overexpressed HEK 293 cells: Expression of PI3Kγ was assessed in control (Sh Vector), stable expression of Sh-RNA targeting PI3Kγ (PI3Kγ KD) and as positive control (+ve) cells stably overexpressing PI3Kγ (PI3Kγ OE). Cell lysates were immunoblotted with anti-PI3Kγ antibody. β-actin was used as loading control. - GRK2 and PI3Kγ interaction with IRS1 and IRS 2: GRK2 has previously been shown to interact with IRS1 and IRS2. To test whether GRK2, PI3Kγ and IRS forms a complex, endogenous IRS1 and IRS2 were immunoprecipitated from plasma membranes of FLAG-β2AR HEK 293 cells stimulated with INS (100 nM) for 10 minutes. Immunoblotting showed co-immunoprecipitation of GRK2 and PI3Kγ, (n=3). - Recruitment of GRK2 in response to INS stimulation in the absence of PI3Kγ: Since absence of PI3Kγ in PI3Kγ KD cells results in abrogation of β2AR phosphorylation with INS, we tested whether GRK recruitment to the plasma membrane still occurs following INS. Immunoblotting for GRK2 in plasma membranes of Sh Vector and PI3Kγ KD HEK 293 cells transfected with FLAG-β2AR following INS stimulation (100 nM) for 10 minutes. (n=4) shows robust recruitment of GRK2 to the plasma membrane in PI3Kγ KD cells and yet we see reduced β2AR phosphorylation consistent with increased β2AR-associated phosphatase activity .

Article Snippet: The membranes were blocked with 5% BSA and immunoblotted using primary anti-phospho-β2AR antibody (1:1000) (Santa Cruz Biotechnology), anti-PI3Kγ (1:500) (Santa Cruz Biotechnology), anti-GRK2 (1:1000) (Santa Cruz Biotechnology), anti-I2PP2A (1:700) (Santa Cruz Biotechnology or) or anti-PP2Ac (1:3000) (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Saline, Control, Expressing, Knockdown, Plasmid Preparation, Positive Control, Stable Transfection, Immunoprecipitation, Clinical Proteomics, Western Blot, Membrane, Transfection, Activity Assay

Journal: bioRxiv

Article Title: Insulin inhibits protein phosphatase 2A to impair β-adrenergic receptor function

doi: 10.1101/2020.09.17.302448

Figure Lengend Snippet: (A) cAMP generation was assessed using luciferase assay following pre-treatment with INS and re-challenged with ISO in FLAG-β2AR HEK 293 cells compared to untreated control cells, *p< 0.001 versus control (Sal), #p< 0.001 versus ISO. (B) β-arrestin (green) recruitment to the plasma membrane was visualized by confocal microscopy using a double stable cell line expressing β2AR and β-Arrestin 2-GFP following Sal, ISO or INS treatment for 10 minutes. Nucleus was visualized by DAPI (blue) staining. Scale bar: 10 μm (C) Immunoblotting for phospho-β2AR upon pre-treatment with propranolol (β-blocker) followed by INS treatment in the FLAG-β2AR HEK 293 cells. FLAG immunoblotting was performed as loading control. Cumulative densitometry shown as bar graph below. (n=5), *p< 0.001 versus control (Sal).

Article Snippet: The membranes were blocked with 5% BSA and immunoblotted using primary anti-phospho-β2AR antibody (1:1000) (Santa Cruz Biotechnology), anti-PI3Kγ (1:500) (Santa Cruz Biotechnology), anti-GRK2 (1:1000) (Santa Cruz Biotechnology), anti-I2PP2A (1:700) (Santa Cruz Biotechnology or) or anti-PP2Ac (1:3000) (Santa Cruz Biotechnology).

Techniques: Luciferase, Control, Clinical Proteomics, Membrane, Confocal Microscopy, Stable Transfection, Expressing, Staining, Western Blot

Journal: bioRxiv

Article Title: Insulin inhibits protein phosphatase 2A to impair β-adrenergic receptor function

doi: 10.1101/2020.09.17.302448

Figure Lengend Snippet: (A) Immunoblotting for GRK2 and PI3Kγ on the plasma membranes of FLAG-β2AR HEK 293 cells following INS stimulation for 10 minutes. Cumulative data for GRK2 and PI3Kγ recruitment. (n=5), *p< 0.001 versus control (Sal). (B) PI3Kγ was immunoprecipitated from plasma membranes of FLAG-β2AR HEK 293 cells stimulated with INS for 10 minutes. Immunoprecipitates were immunoblotted for co-immunoprecipitating phospho-β2AR and GRK2. Densitometric analysis is shown in the bar graph. (n=4). *p < 0.05 compared to control (Sal). (C) Sf 9 baculovirus purified GRK2 protein was immobilized on the CM5 Biacore chip and increasing concentrations of Sf 9 baculovirus purified PI3Kγ was used as analyte. Measurement of dissociation constant shows robust interaction between GRK2-PI3Kγ with a Kd of 60 nM.

Article Snippet: The membranes were blocked with 5% BSA and immunoblotted using primary anti-phospho-β2AR antibody (1:1000) (Santa Cruz Biotechnology), anti-PI3Kγ (1:500) (Santa Cruz Biotechnology), anti-GRK2 (1:1000) (Santa Cruz Biotechnology), anti-I2PP2A (1:700) (Santa Cruz Biotechnology or) or anti-PP2Ac (1:3000) (Santa Cruz Biotechnology).

Techniques: Western Blot, Clinical Proteomics, Control, Immunoprecipitation, Purification

Journal: bioRxiv

Article Title: Insulin inhibits protein phosphatase 2A to impair β-adrenergic receptor function

doi: 10.1101/2020.09.17.302448

Figure Lengend Snippet: (A) FLAG-β2AR immunoprecipitated from plasma membrane fraction of FLAG-β2AR HEK 293 cells and associated phosphatase activity assessed following INS stimulation for 10 minutes. (n=3). *p < 0.001 compared to control (Sal). (B) FLAG-β2AR was transfected into Sh Vector and PI3Kγ KD HEK 293 cells and FLAG-β2AR immunoprecipitated from plasma membranes and assessed for associated phosphatase activity following INS stimulation for 10 minutes, (n=3). *p < 0.001 compared to control (Sal). (C) Immunoblotting was performed to assess phospho-I2PP2A levels in Sh Vector and PI3Kγ KD cells following INS stimulation for 10 minutes. Total I2PP2A was immunoblotted as loading control. Cumulative densitometry is presented in the bar graphs. (n = 5). *p < 0.001 compared to control (Sal). (D) FLAG-β2AR was transfected into HEK 293 parental cells, CRISPR I2PP2A or stable Sh I2PP2A knock down cells and immunoblotting was performed to assess phospho-β2AR, phospho-I2PP2A and I2PP2A in total cell lysates. Cumulative densitometry is presented in the bar graphs. (n = 4). *p < 0.001 compared to control (Sal).

Article Snippet: The membranes were blocked with 5% BSA and immunoblotted using primary anti-phospho-β2AR antibody (1:1000) (Santa Cruz Biotechnology), anti-PI3Kγ (1:500) (Santa Cruz Biotechnology), anti-GRK2 (1:1000) (Santa Cruz Biotechnology), anti-I2PP2A (1:700) (Santa Cruz Biotechnology or) or anti-PP2Ac (1:3000) (Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Clinical Proteomics, Membrane, Activity Assay, Control, Transfection, Plasmid Preparation, Western Blot, CRISPR, Knockdown

Journal: bioRxiv

Article Title: Insulin inhibits protein phosphatase 2A to impair β-adrenergic receptor function

doi: 10.1101/2020.09.17.302448

Figure Lengend Snippet: (A) Immunoblots to assess phospho-β2AR in cardiac lysates from mice infused with Sal or INS for 2 weeks. Cumulative data is presented in the bar graphs. (n = 6). *p < 0.0001 compared to control (Sal). (B) Immunoblotting for phospho-β2AR, GRK2 and PI3Kγ in total cell lysates of primary cardiac myocytes isolated from mice infused with Sal or INS for 2 weeks. (n=6). (C) Immunoblots to assess phospho-β2AR in cardiac lysates from mice injected with Insulin or Saline for 20 minutes. Densitometry is presented in the bar graphs. (n = 6). *p < 0.0001 compared to control (Sal). (D) Immunoblots to assess phospho-β2AR in primary cardiac myocytes isolated from WT and PI3Kγ KO mice and stimulated with INS for 0, 10, 20 and 30 minutes. Cumulative data is presented in the bar graphs. (n = 5). *p < 0.001 compared to control (Sal). (E) Immunoblots to assess phospho-β2AR in primary cardiac fibroblasts isolated from WT and PI3Kγ KO mice and stimulated with INS for 10 minutes. Cumulative densitometry is presented in the bar graphs. (n = 5). *p < 0.001 compared to control (Sal).

Article Snippet: The membranes were blocked with 5% BSA and immunoblotted using primary anti-phospho-β2AR antibody (1:1000) (Santa Cruz Biotechnology), anti-PI3Kγ (1:500) (Santa Cruz Biotechnology), anti-GRK2 (1:1000) (Santa Cruz Biotechnology), anti-I2PP2A (1:700) (Santa Cruz Biotechnology or) or anti-PP2Ac (1:3000) (Santa Cruz Biotechnology).

Techniques: Western Blot, Control, Isolation, Injection, Saline

Journal: Frontiers in Behavioral Neuroscience

Article Title: Lack of β2-AR Increases Anxiety-Like Behaviors and Rewarding Properties of Cocaine

doi: 10.3389/fnbeh.2017.00049

Figure Lengend Snippet: Sequence of behavioral tests. The anxiety level and depression level tests were carried out following the first and second cohorts, respectively. Acute cocaine response and cocaine conditioned place preference (CPP) were tested following the third and fourth cohorts, respectively (the first cohort: wild type (WT) n = 26–27, heterozygous (HET) n = 17, knock-out (KO) n = 27; the second cohort: WT n = 12, HET n = 18, KO n = 16; the third cohort: WT n = 9, HET n = 6, KO n = 9; the forth cohort: WT n = 18, HET n = 8, KO n = 20).

Article Snippet: Then the membranes were incubated in primary antibody for β1-AR (1:100, Santa cruz), β2-AR (1:100, Santa cruz) or β-tubulin (1:2500; Sigma, St Louis, MO, USA) at 4°C overnight.

Techniques: Sequencing, Conditioned Place Preference, Knock-Out

Journal: Frontiers in Behavioral Neuroscience

Article Title: Lack of β2-AR Increases Anxiety-Like Behaviors and Rewarding Properties of Cocaine

doi: 10.3389/fnbeh.2017.00049

Figure Lengend Snippet: Anxiety level was elevated in β2-adrenoceptors (β2-AR) KO mice. (A) Representative images of β2-AR and β1-AR expression in the hippocampus of the WT and β2-AR KO mice by WB analysis and the quantification of β1-AR and β2-AR expression in hippocampus membrane fractions. (B) Representative images of β2-AR and β1-AR expression in the medial prefrontal cortex (mPFC) of the WT and β2-AR KO and the quantification of β1-AR and β2-AR expression in mPFC membrane fractions. (C,D) The anxiety level of WT, HET and β2-AR KO mice. (C) Light/Dark box test WT: n = 27, HET: n = 17, β2-AR KO: n = 27. (D) Elevated plus maze test. WT: n = 26, HET: n = 17, β2-AR KO: n = 27. Data are presented as mean ± SEM * p < 0.05 vs. WT, ** p < 0.01 vs. WT, *** p < 0.001 vs. WT.

Article Snippet: Then the membranes were incubated in primary antibody for β1-AR (1:100, Santa cruz), β2-AR (1:100, Santa cruz) or β-tubulin (1:2500; Sigma, St Louis, MO, USA) at 4°C overnight.

Techniques: Expressing, Membrane

Journal: Frontiers in Behavioral Neuroscience

Article Title: Lack of β2-AR Increases Anxiety-Like Behaviors and Rewarding Properties of Cocaine

doi: 10.3389/fnbeh.2017.00049

Figure Lengend Snippet: Depression-like behavior was suppressed in β2-AR KO mice. The performance of WT, HET and β2-AR KO mice in Tail-suspension (A,B) and Forced swimming (C,D) tests. Total immobility time during 5-min period (A,C) in each 1-min block (B,D) are plotted. For tail-suspension test (TST): WT: n = 12; HET: n = 18; KO: n = 16. For Forced swimming test (FST): WT: n = 12; HET: n = 18; KO: n = 16). Data are presented as mean ± SEM * p < 0.05 vs. WT, ** p < 0.01 vs. HET.

Article Snippet: Then the membranes were incubated in primary antibody for β1-AR (1:100, Santa cruz), β2-AR (1:100, Santa cruz) or β-tubulin (1:2500; Sigma, St Louis, MO, USA) at 4°C overnight.

Techniques: Suspension, Blocking Assay