Journal: The Journal of general virology

Article Title: p38 mitogen-activated protein kinase is crucial for bovine papillomavirus type-1 transformation of equine fibroblasts.

doi: 10.1099/vir.0.031526-0

Figure Lengend Snippet: Fig. 3. Detection of phospho-MAPKAPK2 (p-MK2) expression by immunofluorescence in equine fibroblasts. (a) The cells were co-stained with the primary rabbit anti-p-MPK2 (Thr334) antibody and secondary Alexa Fluor 488 chicken anti-rabbit IgG for p-MK2 and TRITC-conjugated phalloidin for filamentous actin (F-actin). Magnification, 400. (b) Co-immunofluorescence staining of p-MK2 and F-actin in empty vector EqPalF cells (neo) or S6-2 cells using antibodies as in (a). Magnification, 400.

Article Snippet: Primary antibody incubation was carried out overnight at 4 uC with rabbit antibodies against p38 MAPK, phospho-p38 (Thr180/Tyr182) or phospho-MAPKAPK2 (Thr 334) (Cell Signaling Technology) all at 1 : 1000 dilutions.

Techniques: Expressing, Immunofluorescence, Staining, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Calcium-mediated Stress Kinase Activation by DMP1 Promotes Osteoblast Differentiation *

doi: 10.1074/jbc.M110.145607

Figure Lengend Snippet: DMP1 stimulation activates MAPKAPK2 and HSP27 that are downstream targets of the p38 MAPK signaling pathway. Confluent adherent MC3T3-E1 cells were treated without (control, C) or with DMP1, and Western blot analysis was performed with anti-phospho-MAPKAPK2 and total MAPKAPK2. A, phosphorylation of MAPKAPK2 was assessed following DMP1 stimulation. Densitometric quantification of the blots was performed by assessing tubulin and total MAPKAPK2. B, phosphorylation of MAPKAPK2 was assessed following treatment with SB203580 and then stimulated by DMP1. C, Western blot was also performed with anti-phospho-HSP27 antibody after stimulating cells without (control) or with DMP1. Equal amounts of proteins were loaded as assessed by tubulin. D, confocal microscopy images showing nuclear localization of HSP27 in DMP1-stimulated cells. The scale bar indicates 20 μm. MC3T3-E1 cells were transfected with MAPKAPK2 wild type (WT), dominant negative kinase-inactive mutant (DN), or constitutively active mutant (CA) or with empty vector (EV). 48 h after transfection, cells were changed to serum-free media, and 24 h later cells were treated with or without DMP1 for 1 h, and phospho-HSP27 (E), phospho-MAPKAPK2 (F), and tubulin were detected by Western blot analysis.

Article Snippet: After electrophoresis, the proteins were electrotransferred onto nitrocellulose membrane (Bio-Rad), blocked with 5% nonfat milk, probed with either anti-Gα q (1:500) (Santa Cruz Biotechnology), anti-p38 (1:500 (Cell Signaling), anti-phospho-p38 (1:500) (Santa Cruz Biotechnology), anti-phospho-HSP27 (1:500) (Cell Signaling), anti-MAPKAPK2 (1:500) (Santa Cruz Biotechnology), and anti-phospho-MAPKAPK2 (1:500) (Cell Signaling) for 16 h at 4 °C.

Techniques: Western Blot, Confocal Microscopy, Transfection, Dominant Negative Mutation, Mutagenesis, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: MK2 Promotes the Development and Progression of Pancreatic Neuroendocrine Tumors Mediated by Macrophages and Metabolomic Factors

doi: 10.3390/ijms232113561

Figure Lengend Snippet: MK2 is activated in mouse PNETs, and its inhibition improves survival time and suppresses tumor growth in a PNET model . Control pancreas and PNETs were stained for phospho-MK2 ( A ), where was highly expressed as shown by a representative image. The RipTag2 mice were treated as shown in a timeline ( B ) where the sequence of procedures and treatments in the transgenic RipTag2 mice model of PNETs were presented. Furthermore, immunofluorescence staining ( C ) and Kaplan–Meier curve ( D ) show decreased activity of MK2 and increased survival time in MK2 inhibitor-treated mice (MK2i, red), and the weight of PNETs ( E ) were decreased in mice treated with MK2 inhibitor (n = 14 in each group). In PNETs, the gene expression ( F ) of Adgre1 , Casp3 , Fasl and Nos2 are increased while Arg1 is decreased in mice treated with MK2 inhibitor, (n = 6 in each group). PNETs were stained for phospho-MK2 and F4/80 where co-expression of MK2 and macrophage marker was shown by a representative image ( G ). Data are presented as means ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Article Snippet: RipTag2 tumors were fixed in 4% paraformaldehyde for up to 24 h, blocked with 2%rat serum and incubated with commercially available antibodies against phospho-MK2 (p-MK2, cat. sc-101729, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or NOS2-APC780 (cat. 47-5920-82, ThermoFisher Scientific) at 1:200 dilution.

Techniques: Inhibition, Control, Staining, Sequencing, Transgenic Assay, Immunofluorescence, Activity Assay, Gene Expression, Expressing, Marker

Journal: International Journal of Molecular Sciences

Article Title: MK2 Promotes the Development and Progression of Pancreatic Neuroendocrine Tumors Mediated by Macrophages and Metabolomic Factors

doi: 10.3390/ijms232113561

Figure Lengend Snippet: MK2 inhibition affects production and secretion of cytokines and chemokines in PNETs. Tumor supernatants were examined by multiplex array for the concentration of G-CSF ( A ), IL-6 ( B ), IL-10 ( C ), IL-12 ( D ), KC ( E ), LIF ( F ), MCP-1 ( G ), MIP-1α ( H ), MIP-1β ( I ), MIP-2 ( J) and VEGF ( K ) from tumors obtained from RipTag2 mice treated with DMSO (control, blue) and MK2 inhibitor (MK2i, red). Data are presented as means ± SEM; * p < 0.05 vs. control; n = 6 in each group.

Article Snippet: RipTag2 tumors were fixed in 4% paraformaldehyde for up to 24 h, blocked with 2%rat serum and incubated with commercially available antibodies against phospho-MK2 (p-MK2, cat. sc-101729, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or NOS2-APC780 (cat. 47-5920-82, ThermoFisher Scientific) at 1:200 dilution.

Techniques: Inhibition, Multiplex Assay, Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: MK2 Promotes the Development and Progression of Pancreatic Neuroendocrine Tumors Mediated by Macrophages and Metabolomic Factors

doi: 10.3390/ijms232113561

Figure Lengend Snippet: MK2 inhibition suppress production of metabolic factors in PNETs. Tumor supernatants were examined by multiplex array for the concentration of GCG ( A ), GIP ( B ), GLP-1 ( C ), IAPP ( D ), INS ( E ), LEP ( F ), PP ( G ), PYY ( H ) and RETN ( I ) from tumors obtained from RipTag2 mice treated with DMSO (control, blue) and MK2 inhibitor (MK2i, red). Data are presented as means ± SEM; * p < 0.05 vs. control; n = 6 in each group.

Article Snippet: RipTag2 tumors were fixed in 4% paraformaldehyde for up to 24 h, blocked with 2%rat serum and incubated with commercially available antibodies against phospho-MK2 (p-MK2, cat. sc-101729, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or NOS2-APC780 (cat. 47-5920-82, ThermoFisher Scientific) at 1:200 dilution.

Techniques: Inhibition, Multiplex Assay, Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: MK2 Promotes the Development and Progression of Pancreatic Neuroendocrine Tumors Mediated by Macrophages and Metabolomic Factors

doi: 10.3390/ijms232113561

Figure Lengend Snippet: MK2 KO BMMs are anti-tumorigenic in vitro and in vivo. In vitro studies in the transgenic and the RipTag2 cell-derived allograft tumor models were performed ( A ). Tumor cells ( B ) were plated and cultured from a dissociated RipTag2 tumor. Cell supernatants were examined with a multiplex array for the concentration of CPEP, IAPP and INS ( C ), GCG, GIP, GLP-1, RETN and SCT ( D ). BMMs from WT and MK2 KO mice were cultured, and supernatants examined for the level of IL-10 ( E ) and IL-12 ( F ) by multiplex array after incubation with LPS for 24 h. The percent of annexin V + cells of gated tumor cells was analyzed ( G ) in co-cultures, n = 8 in each group. Representative histograms ( H ) of the flow cytometry analysis of annexin V in the RipTag2 cells treated with activated WT and MK2 KO BMMs. Data are presented as means ± SEM; * p < 0.05 vs. WT BMMs + LPS; *** p < 0.001 vs. RipTag2 cells; ### p < 0.001 vs. RipTag2 cells + non. activated WT or MK2 KO BMMs; $$ p < 0.01 vs. RipTag2 cells + activated WT BMMs.

Article Snippet: RipTag2 tumors were fixed in 4% paraformaldehyde for up to 24 h, blocked with 2%rat serum and incubated with commercially available antibodies against phospho-MK2 (p-MK2, cat. sc-101729, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or NOS2-APC780 (cat. 47-5920-82, ThermoFisher Scientific) at 1:200 dilution.

Techniques: In Vitro, In Vivo, Transgenic Assay, Derivative Assay, Cell Culture, Multiplex Assay, Concentration Assay, Incubation, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: MK2 Promotes the Development and Progression of Pancreatic Neuroendocrine Tumors Mediated by Macrophages and Metabolomic Factors

doi: 10.3390/ijms232113561

Figure Lengend Snippet: Adoptive transfer of MK2 KO macrophages improves survival of RipTag2 mice and suppress growth of cell line allograft tumors . The RipTag2 mice were treated with macrophages as shown in a timeline ( A ). The Kaplan–Meier curve ( B ) shows improved survival of mice receiving MK2 KO macrophages compared to WT macrophages, n = 8 in each group. Rag1 KO mice receiving RipTag2 cells were treated with macrophages as presented in the timeline ( C ), and showed decreased tumor growth ( D , E ) with adoptive transfer of MK2 KO macrophages, and these tumors had increased expression of apoptotic genes ( F ) such as Casp3 and Fasl along with decreased expression of Il-10 and increased expression of Il-12 and Nos2 as indicators of macrophage activity, n = 8 in each group. Immunofluorescence staining of NOS2 ( G ) in tumors treated with MK2 KO macrophages shows increased activity of macrophages compared to tumors treated with WT macrophages, n = 11 in each group. Data are presented as means ± SEM; * p < 0.05, *** p < 0.001 vs. control; # p < 0.05, ### p < 0.001 vs. + WT BMMs.

Article Snippet: RipTag2 tumors were fixed in 4% paraformaldehyde for up to 24 h, blocked with 2%rat serum and incubated with commercially available antibodies against phospho-MK2 (p-MK2, cat. sc-101729, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or NOS2-APC780 (cat. 47-5920-82, ThermoFisher Scientific) at 1:200 dilution.

Techniques: Adoptive Transfer Assay, Expressing, Activity Assay, Immunofluorescence, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: MK2 Promotes the Development and Progression of Pancreatic Neuroendocrine Tumors Mediated by Macrophages and Metabolomic Factors

doi: 10.3390/ijms232113561

Figure Lengend Snippet: MK2 KO macrophages inhibit production and secretion of cytokines/chemokines and metabolic factors in PNETs. Tumor supernatants were examined by multiplex array for the concentration of IL-1β ( A ), IL-6 ( B ), IL-10 ( C ), IL-12 ( D ), MCP-1 ( E ), TNF-α ( F ), INS ( G ), LEP ( H ) and RETN ( I ) in media from the RipTag2 cell-derived allograft tumors obtained from mice treated with PBS (control, black), WT BMMs (+WT BMMs, blue) and MK2 KO BMMs (+MK2 KO BMMs, red). Data are presented as means ± SEM; * p < 0.05 vs. control; # p < 0.05, ## p < 0.01 vs. + WT BMMs; n = 8 in each group.

Article Snippet: RipTag2 tumors were fixed in 4% paraformaldehyde for up to 24 h, blocked with 2%rat serum and incubated with commercially available antibodies against phospho-MK2 (p-MK2, cat. sc-101729, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or NOS2-APC780 (cat. 47-5920-82, ThermoFisher Scientific) at 1:200 dilution.

Techniques: Multiplex Assay, Concentration Assay, Derivative Assay, Control