Journal: Respiratory Research

Article Title: Dexamethasone rescues TGF-β1-mediated β 2 -adrenergic receptor dysfunction and attenuates phosphodiesterase 4D expression in human airway smooth muscle cells

doi: 10.1186/s12931-020-01522-w

Figure Lengend Snippet: TGF-β1 attenuates expression of grk2 and grk3 . HASM were treated with TGF-β1 (10 ng/ml, 18 h), total RNA was isolated, and gene expression was assessed by TaqMan qPCR. Expression of grk2 and grk3 was normalized to endogenous β-actin. Data is representative of n = 5–6 different donors as mean ± SEM, * p < 0.05 by Student’s t-test

Article Snippet: The following Taqman primer sets were purchased from Thermo Fisher Scientific: ACTB, actin beta, Hs01060665_g1; GRK2, G protein-coupled receptor kinase 2 Hs00176395_m1; GRK3, G protein-coupled receptor kinase 3, Hs00178266_m1; PDE4D, phosphodiesterase 4D, Hs01579625_m1.

Techniques: Expressing, Isolation, Gene Expression

Journal: Annals of the New York Academy of Sciences

Article Title: Spatial regulation of GPR64/ADGRG2 signaling by β-arrestins and GPCR kinases

doi: 10.1111/nyas.14227

Figure Lengend Snippet: GPCR kinases regulate GPR64 trafficking and signaling. HEK cells were transfected with control or GRK-specific siRNAs. (A) Cleared lysates were used for immunoblotting with anti-GRK-specific antibodies or anti-β-actin antibody as a loading control. Representative blots from three independent experiments are shown. (B) Cells were first transfected with siRNAs as above and then transiently transfected with ΔNTF or P622 plasmids in combination with pCRE-Luc plasmid. After an overnight serum starvation, cells were stimulated with either DMSO (vehicle) or 100 μM P-15 for 5 h and the CRE induction was measured. Data are shown in RLUs and are presented as mean ± SEM from a representative experiment out of three individual experiments performed in triplicate. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. Data were compared with control siRNA with one-way ANOVA with Dunnett’s test. (C) Cells were transfected with control or GRK4-specific siRNA followed by GPR64 plasmids. After an overnight serum starvation, cells were fixed and the HA-tagged receptors on the cell surface were labeled with mouse anti-HA antibody and Alexa Fluor 594–conjugated anti-mouse antibody in nonpermeabilizing condition. DAPI was used for nuclear staining. Magnification 400×, scale bar: 20 μm. (D) Histograms of the fluorescence intensity of surface HA-tagged receptors over the length of yellow lines (in C) are shown. (E) Cells were transfected with either control, GRK4-specific, or a combination of GRK3-specific, GRK4-specific, and GRK5-specific siRNAs followed by GPR64-expressing plasmids. The expression of HA-tagged receptors at the cell surface was measured by ELISA at OD 450 nm. Data were normalized to that of cells transfected with control siRNA and FL plasmid (OD value: 0.49 ± 0.01) and are presented as mean ± SEM from three individual experiments performed in triplicate. ∗P < 0.05, ∗∗∗P < 0.001. Data were compared with control siRNA with one-way ANOVA with Dunnett’s test.

Article Snippet: Antibodies Antibodies were purchased from the following sources: Cell Signaling Technologies (Beverly, MA): rabbit anti-HA (#3724), mouse anti-FLAG (#8146), rabbit anti-GRK2 (#3982), rabbit anti-GRK3 (#80362), rabbit anti-GRK6 (#5878), rabbit anti-β-arrestin1 (#30036), rabbit anti-β-arrestin2 (#3857), and rabbit anti-V5 (#13202); Biolegend (San Diego, CA): mouse anti-HA (#901513); Thermo Fisher Scientific: mouse anti-V5 (# {"type":"entrez-nucleotide","attrs":{"text":"R96025","term_id":"981685","term_text":"R96025"}} R96025 ); Abcam (Cambridge, MA): rabbit anti-GRK4 (#ab182635); Sigma: rabbit anti-β-actin (#A2066); RnD Systems (Minneapolis, MN): goat anti-GRK5 (#AF4539).

Techniques: Transfection, Western Blot, Plasmid Preparation, Labeling, Staining, Fluorescence, Expressing, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Suitability of GRK Antibodies for Individual Detection and Quantification of GRK Isoforms in Western Blots

doi: 10.3390/ijms23031195

Figure Lengend Snippet: List of eight commercially available antibodies examined in this study, targeting the ubiquitously expressed human GRK isoforms. Overview of the tested antibodies (monoclonal (mc), polyclonal (pc)) and the tested GRK isoform is provided, including the supplier’s information, our review of each antibody, and the dilution used in Western blot to determine the antibody specificity. Additionally, the number of amino acids (aa) and the calculated approximate molecular weight (MW) for each human GRK isoform are listed. Accession numbers for protein isoforms can be found in the Materials and Methods section and additionally summarized in Supplementary Table S1 .

Article Snippet: GRK3-1 , 688 aa 80 kDa , sc-365197 (C-11) mouse mc , Santa Cruz Biotechnology , raised against amino acids 646-688 of human GRK3 , human, mouse, rat , 1:250 , does not detect overexpressed human GRK3 isoforms—only background band.

Techniques: Western Blot, Molecular Weight

Journal: Science signaling

Article Title: Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

doi: 10.1126/scisignal.aau8072

Figure Lengend Snippet: (A) HEK293 cells stably expressing the hNOP receptor were preincubated with either vehicle (DMSO; control) or the GRK2/3-specific inhibitor compound 101 (cmpd 101) at 30 μM for 30 min at 37 °C, then exposed to 10 μM N/OFQ, 10 μM forskolin, or 1 μM PMA (or not, -) for 10 min. Lysates were then immunoblotted with antibodies to pSer346, pSer351, or pThr362/Ser363. Blots were stripped and reprobed for NOPR. Blots are representative, n=3. (B) Cells described in (A) were preincubated with vehicle (DMSO; (–) or cmpd101 at the indicated concentrations for 30 min at 37 °C, then treated with water (–) or 10 μM N/OFQ for 10 min at 37 °C. Lysates were blotted as described in (A). Blots are representative, n=4. (C and D) Cells described in (A) were transfected with either siRNA targeting GRK2, GRK3, or GRK2 and GRK3 (GRK2/3) or a control (SCR) for 72 hours (C) or with GRK2 or GRK3 expression plasmids or an empty vector (MOCK) for 48 hours (D), then stimulated with 10 μM N/OFQ for 10 min at 37 °C. Lysates were immunoblotted with antibody to pThr362/Ser363. Blots were stripped and reprobed for NOPR. Densitometry, above the blots, was normalized to those in SCR- or MOCK-transfected cells, which were set to 100%. Data are mean ± SEM from five to six independent experiments. *P<0.05 vs. SCR or MOCK by two-way ANOVA with Bonferroni post-test.

Article Snippet: Human GRK2 and GRK3 plasmids were obtained from OriGene and TransOMIC, respectively.

Techniques: Stable Transfection, Expressing, Control, Transfection, Plasmid Preparation