Journal: Cell Communication and Signaling : CCS

Article Title: Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84

doi: 10.1186/s12964-020-0516-2

Figure Lengend Snippet: Effect of dynasore and sucrose on HCA 3 and GPR84 cAMP inhibitory and ERK signaling. a Scheme summarizing all inhibitors and their targets used in the present study. b, c CHO-K1 cells were transiently transfected with HCA 3 or GPR84. b 80 μM dynasore reduced cAMP inhibitory signaling of HCA 3 upon 3HO and 3HDec and of GPR84 upon C10 but not 3HDec stimulation. Sucrose (0.4 M) significantly inhibited only the 3HDec-induced HCA 3 -mediated reduction of intracellular cAMP levels. cAMP level of HCA 3 - or GPR84-transfected cells in absence of agonist is set to 100%, respectively. c The agonist-induced phosphorylation of ERK1/2 was measured in absence and presence of dynasore or sucrose. All agonists induced an increase in pERK/total ERK levels upon stimulation of HCA 3 and GPR84, respectively. Dynasore blocked the signal of 100 μM 3HO and 100 μM 3HDec completely and sucrose partially in HCA 3 –transfected cells. Dynasore did not fully block the signal of 100 μM C10 and 100 μM 3HDec in GPR84-transfected cells. The residual ERK signals of GPR84 in presence of 3HDec and dynasore or sucrose did not differ. pERK/total ERK of HCA 3 - or GPR84-transfected cells in absence of agonist is set 1. b, c Data is given as mean ± SEM of at least three independent experiments each carried out in triplicates. * P ≤ 0.05; ** P ≤ 0.01

Article Snippet: pErk/total Erk content of cell extracts was determined by the Alpha SureFire Ultra Multiplex p-ERK 1/2 + Total ERK assay according to the manufacturer’s protocol (Perkin Elmer LAS).

Techniques: Transfection, Blocking Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84

doi: 10.1186/s12964-020-0516-2

Figure Lengend Snippet: Effect of dyn-2 mutants on HCA 3 and GPR84 cell surface expression, cAMP inhibitory signaling and ERK activation. a-c CHO-K1 cells were transiently co-transfected with HCA 3 or GPR84 and dyn-2 wt, dyn-2 K44A or R399A mutants. a In comparison to dyn-2 wt co-transfected cells HCA 3 and GPR84 cell surface expression was significantly reduced when K44A or R399A were co-transfected. b Basal activity of HCA 3 but not GPR84 was diminished in presence of K44A. Agonist-induced (HCA 3 : 6.25 μM 3HO, 25 μM 3HDec; GPR84: 100 μM C10, 25 μM 3HDec) inhibition of forskolin-stimulated cAMP accumulation was reduced in presence of K44A compared to dyn-2 wt whereas R399A did not affect cAMP inhibitory signaling. c Agonist-induced increase of pERK/total ERK level of HCA 3 (25 μM 3HO, 100 μM 3HDec) and GPR84 (25 μM C10, 25 μM 3HDec) was reduced in presence of K44A and R399A compared to dyn-2 wt. a-c Data is given as mean ± SEM of at least three independent experiments each carried out in triplicates. * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001 ( d ) Images of HEK293-T cells transiently co-expressing HCA 3 -mRuby (red) or GPR84-mRuby and dyn-2-YFP variants (green). In presence of dyn-2 wt, HCA 3 was detected intracellularly and at the plasma membrane where it co-localized with dyn-2 wt. In case of co-expression of HCA 3 with the dyn-2 mutants K44A and R399A, co-localization was detected in perinuclear vesicles as well as certain areas at the plasma membrane. GPR84 was in presence of all dyn-2 variants found mostly at the plasma membrane

Article Snippet: pErk/total Erk content of cell extracts was determined by the Alpha SureFire Ultra Multiplex p-ERK 1/2 + Total ERK assay according to the manufacturer’s protocol (Perkin Elmer LAS).

Techniques: Expressing, Activation Assay, Transfection, Comparison, Activity Assay, Inhibition, Membrane

Journal: Cell Communication and Signaling : CCS

Article Title: Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84

doi: 10.1186/s12964-020-0516-2

Figure Lengend Snippet: Role of β-arrestin-2 for HCA 3 and GPR84 signaling and effect of methyl-β-cyclodextrin (MβCD). a, b CHO-K1 cells were transiently transfected with HCA 3 or GPR84. a MβCD inhibited both, the 3HO- and 3HDec-induced reduction of forskolin (fsk)-induced cAMP levels in HCA 3 -transfected cells. For GPR84, only the C10-induced but not the 3HDec-induced decrease in cAMP was inhibited. Barbardin (100 µM) inhibited only the 3HO-induced HCA 3 -mediated reduction of cAMP levels. cAMP levels of HCA 3 - or GPR84-transfected cells in absence of agonist are set 100%, respectively. b 3 mM MβCD did not affect HCA 3 -mediated activation of ERK by 3HO, but caused a decrease of the signal induced by 100 μM 3HDec. Presence of MβCD caused a decrease in C10-induced GPR84-mediated ERK activation and had no effect on the 3HDec-induced ERK activation. The HCA 3 -mediated activation of ERK by 3HO, but not 3HDec, was inhibited in presence of barbardin. Barbardin had no effect on the GPR84-mediated activation of ERK by 3HDec and C10. pERK/total ERK of HCA 3 - or GPR84-transfected cells in absence of agonist is set 1, respectively. c Live-cell images of HEK293-T cells co-expressing HCA 3 -mRuby (red) and β-arrestin-2-YFP (green) were acquired before stimulation and 30 min post-stimulation with 100 μM 3HO or 100 μM 3HDec. d HEK293-T cells stably expressing β-arrestin-2-EA cells transiently transfected with HCA 3 were stimulated with 3HO and 3HDec. Quantification of β-arrestin-2 recruitment using the PathHunter β-arrestin assay (Eurofins DiscoverX) showed recruitment of β-arrestin-2 by HCA 3 following 3HO but not 3HDec stimulation. Luminescence of HCA 3 or empty vector transfected cells in absence of agonist is set 1, respectively. a, b, d Data is given as mean ± SEM of at least three independent experiments each carried out in triplicates. # P ≤ 0.1* P ≤ 0.05; ** P ≤ 0.01

Article Snippet: pErk/total Erk content of cell extracts was determined by the Alpha SureFire Ultra Multiplex p-ERK 1/2 + Total ERK assay according to the manufacturer’s protocol (Perkin Elmer LAS).

Techniques: Transfection, Activation Assay, Expressing, Stable Transfection, PathHunter β-Arrestin Assay, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84

doi: 10.1186/s12964-020-0516-2

Figure Lengend Snippet: Effect of gallein, an inhibitor of Gβγ subunits, on agonist-induced reduction of cAMP levels and ERK activation of HCA 3 and GPR84. CHO-K1 cells were transiently transfected with HCA 3 or GPR84. a The HCA 3 -mediated reduction of forskolin (fsk)-induced cAMP levels induced by both, 3HO and 3HDec, was significantly diminished in presence of 50 μM gallein. The GPR84-induced decrease in cAMP levels in presence of gallein was only reduced in case of activation by C10 but not 3HDec. cAMP level of HCA 3 - or GPR84-transfected cells in absence of agonist is set 100%, respectively. b Gallein inhibited the 3HDec-induced HCA 3 -mediated increase in pERK/total ERK levels completely but the 3HO-induced increase only partially. GPR84-mediated activation of ERK by both, C10 and 3HDec, was equally diminished in presence of gallein. pERK/total ERK of HCA 3 - or GPR84-transfected cells in absence of agonist is set 1, respectively. a, b Data is given as mean ± SEM of at least three independent experiments each carried out in triplicates. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001

Article Snippet: pErk/total Erk content of cell extracts was determined by the Alpha SureFire Ultra Multiplex p-ERK 1/2 + Total ERK assay according to the manufacturer’s protocol (Perkin Elmer LAS).

Techniques: Activation Assay, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84

doi: 10.1186/s12964-020-0516-2

Figure Lengend Snippet: Components involved in HCA 3 and GPR84 signal transduction. a, b Agonist-induced phosphorylation of endogenous ERK1/2 in cellular lysates of HCA 3 or GPR84 transfected CHO-K1 cells in absence and presence of 25 μM ZA (zoledronic acid - inhibitor of ras/rho), 100 μM NSC23766 (inhibitor of rac1) and 25 μM Ly294002 (inhibitor of PI3K) was determined. a ZA, NSC23766 and Ly294002 partially inhibited the HCA 3 -induced ERK activation of both agonists. ZA and Ly 294,002 caused a significant reduction of the GPR84-mediated ERK activation by C10, whereas the ERK activation by 3HDec was only affected by presence of Ly294002. NSC23766 did not inhibit the GPR84-induced activation of ERK by either agonist. b Both, the 3HO- and 3HDec-induced ERK activation of HCA 3 did not persist upon removal of agonist. The GPR84-mediated activation of ERK by 3HDec persisted, whereas the C10-induced activation was almost completely diminished 10 min past agonist removal. a, b pERK/total ERK of HCA 3 - or GPR84-transfected cells in absence of agonist is set 1, respectively. Data is given as mean ± SEM of at least three independent experiments each carried out in triplicates. # P ≤ 0.1; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. c 3HO- and 3HDec-induced cAMP inhibitory signaling of HCA 3 was dependent on Gαi, Gβγ subunits and dyn (internalization). Signaling components involved in HCA 3 -mediated ERK activation by 3HO an 3HDec included Gβγ subunits, PI3K, rac1 and ras/rho. HCA 3 activation by 3HO led to β-arrestin-2 recruitment, which was not the case for 3HDec. ERK signaling of HCA 3 by 3HO involved clathrin and by 3HDec caveolin. GPR84 activation by C10 was dependent on Gαi, Gβγ subunits, dyn (internalization), caveolin, ras/rho and PI3K. In contrast, 3HDec-induced cAMP inhibitory signaling was not dependent on Gβγ subunits, dyn, caveolin or clathrin, thus internalization. ERK activation induced by GPR84 upon 3HDec stimulation persisted upon agonist removal and involved PI3K

Article Snippet: pErk/total Erk content of cell extracts was determined by the Alpha SureFire Ultra Multiplex p-ERK 1/2 + Total ERK assay according to the manufacturer’s protocol (Perkin Elmer LAS).

Techniques: Transduction, Transfection, Activation Assay

Journal: Journal of Advanced Research

Article Title: Efficient improvement of the proliferation, differentiation, and anti-arthritic capacity of mesenchymal stem cells by simply culturing on the immobilized FGF2 derived peptide, 44-ERGVVSIKGV-53

doi: 10.1016/j.jare.2023.09.041

Figure Lengend Snippet: Antibody list.

Article Snippet: Membrane blocking was performed using 5 % skimmed milk in Tris-buffered saline for 1 h, followed by incubation with the appropriate primary antibodies against total ERK (Cat No. B7074, Assay Biotechnology, Fremont, CA, USA), phosphorylated ERK (Cat No. 9101 s, Cell Signaling Technology, Danvers, MA, USA), total AKT (Cat No. CSB-PA000855, CUSABIO, Houston, TX, USA), phosphorylated AKT (Cat No. CSB-PA008120, CUSABIO), IKKα (Cat No. 11930, Cell Signaling Technology), FGFR1 (Cat No. NBP2-33784, Novus Biologicals, Centennial, CO, USA), phosphorylated FGFR1 (Tyr653, Tyr654) (Cat No. 44-1140G, Invitrogen) polyclonal Antibody, FRS2 antibody (Cat No. 3836–100, BioVision, Waltham, MA, USA), phosphorylated FRS2-alpha (Cat No. 3864S, Cell Signaling) or ACTIN (Cat No. sc-8432, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4 °C.

Techniques: Staining, Plasmid Preparation

Journal: Frontiers in Molecular Neuroscience

Article Title: The Alpha Isoform of Heat Shock Protein 90 and the Co-chaperones p23 and Cdc37 Promote Opioid Anti-nociception in the Brain

doi: 10.3389/fnmol.2019.00294

Figure Lengend Snippet: Hsp90α regulates opioid signal transduction in the brain. Male CD-1 mice had vehicle or 0.1 nmol KUNA115 injected icv , 24 h, followed by vehicle or 0.1 nmol DAMGO icv , 10 min. Periaqueductal gray region analyzed by Western blot. All data reported as the mean ± SEM, with sample sizes of mice/group noted in each graph; all experiments performed in four technical replicates. For ERK, JNK and Akt, data analyzed by two-way ANOVA with Fisher’s Least Significant Difference post hoc test; **,***,**** p < 0.01, 0.001, 0.0001 vs. Vehicle:Vehicle group. For Hsp70 and STAT3 data analyzed by Unpaired 2-Tailed t -test; * p < 0.05 vs. same target Vehicle group. (A) Representative sample blots shown for each target, with MW indicated for each protein. Each pair of images for one target (e.g., p-Akt) were from the same blot, but discontinuous, so they are separated to denote this fact. (B) All Western data quantitated by target. ERK, JNK, and Akt are phosphorylated protein signal normalized to total protein. Hsp70 and STAT3 are normalized to GAPDH. KUNA115 treatment caused a loss of ERK and JNK stimulation over baseline by DAMGO, and a loss of STAT3 protein expression.

Article Snippet: We used the following antibodies for our Western analysis: phospho-Akt (#50-191-224, Fisher Scientific); total-Akt (#50-190-279, Fisher Scientific); phospho-ERK (#50-191-932, Fisher Scientific); total-ERK (#50-191-129, Fisher Scientific); phospho-JNK (#9255, Cell Signaling); total-JNK (#9252, Cell Signaling); Hsp70 (#4872, Cell Signaling); STAT3 (#9139, Cell Signaling); GAPDH (#PIMA515738, Fisher Scientific); Hsp90α (#MA110892, Fisher Scientific); and mCherry (#NBP196752SS, Fisher Scientific).

Techniques: Transduction, Injection, Western Blot, Expressing

Journal: Aging (Albany NY)

Article Title: Non-genomic mechanisms mediate androgen-induced PSD95 expression

doi: 10.18632/aging.101913

Figure Lengend Snippet: T-BSA upregulated the phosphorylation level of Erk1/2 and eIF4E through ZIP9/Gnα11. ( A – C ) The ratio of phosphorylated/total levels of P38 ( A ), JNK ( B ), and Erk1/2 ( C ) detected in HT22 cells pre-treated with nc-shRNA or Gnα11-shRNA using Flowmetric Luminex xMAP ® assay (n=5). ( D – F ) Western blotting for the phosphorylated/total levels of Erk1/2 and eIF4E induced by T-BSA pre-treated with nc-shRNA, ZIP9-shRNA, or Gnα11-shRNA (n=5). ( G – J ) Immunofluorescence staining for the phosphorylation level of Erk1/2 and eIF4E induced by T-BSA pre-treated with nc-shRNA, ZIP9-shRNA or Gnα11-shRNA (n=4, scale bars = 20 μm). (n.s.: non-significant; * P < 0.05; ** P < 0.01).

Article Snippet: Intracellular Bead-Based Multiplex Assays (cat#: 48-619MAG, 2-Plex Phospho/Total Erk Magnetic bead kit; cat#: 48-624MAG, 2-Plex Phospho/Total p38 Magnetic Bead Kit; cat#: 48-622MAG, 2-Plex Phospho/Total JNK Magnetic Bead Kit; cat#: 46-713MAG, Total β-Tubulin Magnetic Bead MAPmateTM, Merck Millipore, USA) with Luminex technology.

Techniques: Phospho-proteomics, shRNA, Luminex, Western Blot, Immunofluorescence, Staining