Journal: The American Journal of Pathology

Article Title: Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation

doi: 10.1016/j.ajpath.2017.11.017

Figure Lengend Snippet: Antibodies Used in This Study

Article Snippet: Western blots were quantified by densitometry using the ImageJ densitometry program (NIH). table ft1 table-wrap mode="anchored" t5 caption a7 Name Citation (PubMed ID) Supplier (location) Catalog number Clone number Anti-AKAP12 antibody 25188285 Abcam (Cambridge, MA) ab49849 JP74 Antiphosphoserine antibody 19383294 Abcam ab9332 Polyclonal IgG Antiphosphotyrosine antibody 26310847 Abcam ab9319 Polyclonal IgG Anti–α-smooth muscle actin antibody 20043323 Abcam ab5694 Polyclonal IgG Anti-PKCα antibody Genetex (Irvine, CA) GTX130453 Polyclonal IgG Anti–cyclin D1 antibody 25956904 Abcam Ab10540 DCS-6 Anti–collagen Iα (1) antibody 25294683 Novus Biologicals (Littleton, CO) NB600-450 COL-1 Anti–heat shock protein 47 antibody 10811611 Novus Biologicals MAB9166-100 950806 Anti–phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) antibody 28220783 Cell Signaling Technologies (Danvers, MA) 9101 Polyclonal IgG Anti–β-actin−peroxidase antibody (control for in vitro experiments) 27831566 Sigma (St. Louis, MO) A3854 AC-15 Anti-GAPDH antibody (control for in vivo experiments) 28761447 Cell Signaling Technologies 2118 14C10 Anti-desmin antibody 23308088 Dako (Agilent; Santa Clara, CA) M0760 D33 Open in a separate window AKAP, A-kinase anchoring protein; ERK, extracellular signal–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ID, identification; MAPK, mitogen-activated protein kinase; PKC, protein kinase C. Antibodies Used in This Study Coimmunoprecipitation Coimmunoprecipitation of proteins from cells or tissue extracts was performed, as published by us previously.

Techniques: In Vitro, In Vivo

Journal: The American Journal of Pathology

Article Title: Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation

doi: 10.1016/j.ajpath.2017.11.017

Figure Lengend Snippet: Proteins Cross-Linked to AKAP12 in Normal HSCs

Article Snippet: Western blots were quantified by densitometry using the ImageJ densitometry program (NIH). table ft1 table-wrap mode="anchored" t5 caption a7 Name Citation (PubMed ID) Supplier (location) Catalog number Clone number Anti-AKAP12 antibody 25188285 Abcam (Cambridge, MA) ab49849 JP74 Antiphosphoserine antibody 19383294 Abcam ab9332 Polyclonal IgG Antiphosphotyrosine antibody 26310847 Abcam ab9319 Polyclonal IgG Anti–α-smooth muscle actin antibody 20043323 Abcam ab5694 Polyclonal IgG Anti-PKCα antibody Genetex (Irvine, CA) GTX130453 Polyclonal IgG Anti–cyclin D1 antibody 25956904 Abcam Ab10540 DCS-6 Anti–collagen Iα (1) antibody 25294683 Novus Biologicals (Littleton, CO) NB600-450 COL-1 Anti–heat shock protein 47 antibody 10811611 Novus Biologicals MAB9166-100 950806 Anti–phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) antibody 28220783 Cell Signaling Technologies (Danvers, MA) 9101 Polyclonal IgG Anti–β-actin−peroxidase antibody (control for in vitro experiments) 27831566 Sigma (St. Louis, MO) A3854 AC-15 Anti-GAPDH antibody (control for in vivo experiments) 28761447 Cell Signaling Technologies 2118 14C10 Anti-desmin antibody 23308088 Dako (Agilent; Santa Clara, CA) M0760 D33 Open in a separate window AKAP, A-kinase anchoring protein; ERK, extracellular signal–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ID, identification; MAPK, mitogen-activated protein kinase; PKC, protein kinase C. Antibodies Used in This Study Coimmunoprecipitation Coimmunoprecipitation of proteins from cells or tissue extracts was performed, as published by us previously.

Techniques: Sequencing

Journal: The American Journal of Pathology

Article Title: Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation

doi: 10.1016/j.ajpath.2017.11.017

Figure Lengend Snippet: Ethanol exposure inhibits the interaction of AKAP12 with the collagen chaperone, heat shock protein 47 (HSP47), in hepatic stellate cells (HSCs). A: Control or ethanol-treated HSCs were immunoprecipitated (IP) with AKAP12 or collagen and then Western blotted (WB) for HSP47 or collagen (Materials and Methods). B: IP blot: Phosphorylated and unphosphorylated fractions of control or ethanol-treated HSCs were purified using Qiagen columns (Materials and Methods) and subjected to immunoprecipitation with HSP47 antibody, followed by Western blotting for AKAP12. Phos-tag blot: AKAP12 phosphorylated (P-AKAP) and unphosphorylated Qiagen fractions from control or ethanol-treated HSCs were compared on Phos-tag gels. Phosphorylated extracellular signal–regulated kinase (p-ERK) was a positive control and is found mainly in the phosphorylated fraction. C: Control or ethanol-fed mouse livers were immunoprecipitated with HSP47 antibody followed by Western blotting for AKAP12. Three experiments are shown. D: Normal or alcoholic hepatitis liver extracts were immunoprecipitated with HSP47 antibody, followed by Western blotting for AKAP12. Experiments were performed in duplicate. Data are expressed as means ± SEM (A–D); n = 5 experiments (A and C); n = 3 experiments (B and D). ∗P < 0.05, ∗∗P < 0.01 versus control; †P < 0.05, ††P < 0.01, and ††††P < 0.001 versus unphosphorylated control; ‡‡‡‡P < 0.001 versus pair-fed control; §§§§P < 0.001 versus normal. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Western blots were quantified by densitometry using the ImageJ densitometry program (NIH). table ft1 table-wrap mode="anchored" t5 caption a7 Name Citation (PubMed ID) Supplier (location) Catalog number Clone number Anti-AKAP12 antibody 25188285 Abcam (Cambridge, MA) ab49849 JP74 Antiphosphoserine antibody 19383294 Abcam ab9332 Polyclonal IgG Antiphosphotyrosine antibody 26310847 Abcam ab9319 Polyclonal IgG Anti–α-smooth muscle actin antibody 20043323 Abcam ab5694 Polyclonal IgG Anti-PKCα antibody Genetex (Irvine, CA) GTX130453 Polyclonal IgG Anti–cyclin D1 antibody 25956904 Abcam Ab10540 DCS-6 Anti–collagen Iα (1) antibody 25294683 Novus Biologicals (Littleton, CO) NB600-450 COL-1 Anti–heat shock protein 47 antibody 10811611 Novus Biologicals MAB9166-100 950806 Anti–phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) antibody 28220783 Cell Signaling Technologies (Danvers, MA) 9101 Polyclonal IgG Anti–β-actin−peroxidase antibody (control for in vitro experiments) 27831566 Sigma (St. Louis, MO) A3854 AC-15 Anti-GAPDH antibody (control for in vivo experiments) 28761447 Cell Signaling Technologies 2118 14C10 Anti-desmin antibody 23308088 Dako (Agilent; Santa Clara, CA) M0760 D33 Open in a separate window AKAP, A-kinase anchoring protein; ERK, extracellular signal–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ID, identification; MAPK, mitogen-activated protein kinase; PKC, protein kinase C. Antibodies Used in This Study Coimmunoprecipitation Coimmunoprecipitation of proteins from cells or tissue extracts was performed, as published by us previously.

Techniques: Immunoprecipitation, Western Blot, Purification, Positive Control

Journal: The American Journal of Pathology

Article Title: Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation

doi: 10.1016/j.ajpath.2017.11.017

Figure Lengend Snippet: AKAP12 regulates the ability of collagen to interact with heat shock protein 47 (HSP47). A: Hepatic stellate cells (HSCs) transfected with AKAP12 or negative control siRNA immunoprecipitated (IP) with collagen antibody, followed by HSP47 immunoblotting. B: HSCs transfected with AKAP12 vector or empty vector control were immunoprecipitated with collagen antibody, followed by HSP47 Western blotting (WB). Data are expressed as means ± SEM (A and B). n = 5 experiments (A and B). ∗P < 0.05, ∗∗∗∗P < 0.001 versus negative control; ††P < 0.01, ††††P < 0.001 versus empty vector.

Article Snippet: Western blots were quantified by densitometry using the ImageJ densitometry program (NIH). table ft1 table-wrap mode="anchored" t5 caption a7 Name Citation (PubMed ID) Supplier (location) Catalog number Clone number Anti-AKAP12 antibody 25188285 Abcam (Cambridge, MA) ab49849 JP74 Antiphosphoserine antibody 19383294 Abcam ab9332 Polyclonal IgG Antiphosphotyrosine antibody 26310847 Abcam ab9319 Polyclonal IgG Anti–α-smooth muscle actin antibody 20043323 Abcam ab5694 Polyclonal IgG Anti-PKCα antibody Genetex (Irvine, CA) GTX130453 Polyclonal IgG Anti–cyclin D1 antibody 25956904 Abcam Ab10540 DCS-6 Anti–collagen Iα (1) antibody 25294683 Novus Biologicals (Littleton, CO) NB600-450 COL-1 Anti–heat shock protein 47 antibody 10811611 Novus Biologicals MAB9166-100 950806 Anti–phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) antibody 28220783 Cell Signaling Technologies (Danvers, MA) 9101 Polyclonal IgG Anti–β-actin−peroxidase antibody (control for in vitro experiments) 27831566 Sigma (St. Louis, MO) A3854 AC-15 Anti-GAPDH antibody (control for in vivo experiments) 28761447 Cell Signaling Technologies 2118 14C10 Anti-desmin antibody 23308088 Dako (Agilent; Santa Clara, CA) M0760 D33 Open in a separate window AKAP, A-kinase anchoring protein; ERK, extracellular signal–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ID, identification; MAPK, mitogen-activated protein kinase; PKC, protein kinase C. Antibodies Used in This Study Coimmunoprecipitation Coimmunoprecipitation of proteins from cells or tissue extracts was performed, as published by us previously.

Techniques: Transfection, Negative Control, Immunoprecipitation, Western Blot, Plasmid Preparation

Journal: The American Journal of Pathology

Article Title: Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation

doi: 10.1016/j.ajpath.2017.11.017

Figure Lengend Snippet: Summary of mechanisms of action of phospho-AKAP12 in alcoholic liver injury. In normal liver, AKAP12 acts as a scaffold protein for protein kinase C (PKCα) and cyclin-D1 (CCND1). Both hepatocytes and hepatic stellate cells (HSCs) exhibit these functions. Normal HSCs also exhibit specific interaction of AKAP12 with the collagen chaperone, heat shock protein 47 (HSP47), and through this interaction, they might regulate collagen maturation and secretion. During alcoholic liver injury, there is decreased interaction of AKAP12 with PKCα and CCND1 in HSCs because of enhanced AKAP12 phosphorylation and loss of its scaffolding activity. Loss of this scaffolding activity may result in changes in HSC proliferation, cytokinesis, and altered signaling. HSC-specific scaffolding of HSP47 is lost when AKAP12 is phosphorylated on alcohol exposure, and this may facilitate HSP47-collagen chaperoning that may further induce collagen maturation and secretion.

Article Snippet: Western blots were quantified by densitometry using the ImageJ densitometry program (NIH). table ft1 table-wrap mode="anchored" t5 caption a7 Name Citation (PubMed ID) Supplier (location) Catalog number Clone number Anti-AKAP12 antibody 25188285 Abcam (Cambridge, MA) ab49849 JP74 Antiphosphoserine antibody 19383294 Abcam ab9332 Polyclonal IgG Antiphosphotyrosine antibody 26310847 Abcam ab9319 Polyclonal IgG Anti–α-smooth muscle actin antibody 20043323 Abcam ab5694 Polyclonal IgG Anti-PKCα antibody Genetex (Irvine, CA) GTX130453 Polyclonal IgG Anti–cyclin D1 antibody 25956904 Abcam Ab10540 DCS-6 Anti–collagen Iα (1) antibody 25294683 Novus Biologicals (Littleton, CO) NB600-450 COL-1 Anti–heat shock protein 47 antibody 10811611 Novus Biologicals MAB9166-100 950806 Anti–phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) antibody 28220783 Cell Signaling Technologies (Danvers, MA) 9101 Polyclonal IgG Anti–β-actin−peroxidase antibody (control for in vitro experiments) 27831566 Sigma (St. Louis, MO) A3854 AC-15 Anti-GAPDH antibody (control for in vivo experiments) 28761447 Cell Signaling Technologies 2118 14C10 Anti-desmin antibody 23308088 Dako (Agilent; Santa Clara, CA) M0760 D33 Open in a separate window AKAP, A-kinase anchoring protein; ERK, extracellular signal–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ID, identification; MAPK, mitogen-activated protein kinase; PKC, protein kinase C. Antibodies Used in This Study Coimmunoprecipitation Coimmunoprecipitation of proteins from cells or tissue extracts was performed, as published by us previously.

Techniques: Scaffolding, Activity 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 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: Experimental and Therapeutic Medicine

Article Title: Aspirin suppresses TNF-α-induced MMP-9 expression via NF-κB and MAPK signaling pathways in RAW264.7 cells

doi: 10.3892/etm.2017.5252

Figure Lengend Snippet: Effects of aspirin on TNF-α-stimulated activation of MAPK signaling pathway in RAW264.7 cells. (A) Aspirin inhibited the TNF-α-stimulated phosphorylation levels of ERK1/2, p38 MAPK and JNK, as determined using western blot analysis. Cells were incubated for 1 h in the absence or present of aspirin (600 µM) and then stimulated for 10, 20, 30 and 60 min with 10 ng/ml of TNF-α. (B) Reverse transcription-quantitative polymerase chain reaction and (C) western blot analysis were performed to examine the effect of MAPK inhibitors on the mRNA and protein expression levels of MMP-9, respectively. Cells were pre-incubated with or without 10 µM PD98059 (p-ERK inhibitor), 10 µM SB203580 (p-p38 inhibitor), SP600125 (p-JNK inhibitor) and aspirin (600 µM) for 1 h and then with TNF-α (10 ng/ml) for 24 h. Densitometric results are represented the mean ± standard deviation of three independent measurements. #P<0.05 and ##P<0.01 vs. untreated control; *P<0.05 and **P<0.01 vs. TNF-α treatment alone. TNF-α, tumor necrosis factor-α; MMP-9, matrix metalloproteinase-9; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; p-, phosphorylated.

Article Snippet: Antibodies against JNK (1:500 dilution, BS6448), p38 (1:500 dilution, BS3566), ERK (1:1,000 dilution, AP0485), phospho-JNK (1:500 dilution, BS4763), phospho-p38 (1:500 dilution, BS4635) and phospho-ERK (1:1,000 dilution, BS4759) were purchased from Bioworld Technology (Beijing, China).

Techniques: Activation Assay, Western Blot, Incubation, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation