Journal: Pharmacological research

Article Title: Degradation of MK2 with natural compound andrographolide: A new modality for anti-inflammatory therapy.

doi: 10.1016/j.phrs.2023.106861

Figure Lengend Snippet: Fig. 2. AG downregulates MK2 protein levels and blocks MK2-mediated inflammatory responses in macrophages. A. Effect of AG on members of the MAPK axis and PAM pathway. RAW264.7 cells were incubated with increasing concentrations of AG (i.e., 5, 10, 20, 50 μM) for 4 h. Cell lysates were analyzed by immunoblotting (n = 3). B. Effect of AG on MK2 protein levels. Band intensities were analyzed by ImageJ, and the MK2/GAPDH ratios relative to untreated cells are indicated. Data are mean, n = 3. (C–D) Effects of AG on LPS-induced mRNA and protein levels of TNF-α and MCP-1. C. RAW264.7 cells were pre-incubated with increasing concentrations of AG for 1 h and then treated with LPS (100 ng/mL) for 4 h. Quantitative PCR was performed. Data are mean ± SEM, n = 3. D. RAW264.7 cells were pre-incubated with increasing concentrations of AG for 1 h and then treated with LPS (100 ng/mL). Cell supernatants were assayed by ELISA (4 h for TNF-α and 24 h for MCP-1 after LPS challenge). Data are mean ± SEM, n = 4. E. Effect of AG (10 μM) on MK2 protein levels in primary AMs with or without LPS (100 ng/ mL) challenge for 4 h. Cell lysates were analyzed by immunoblotting (n = 3). F. Effects of AG on primary AM inflammatory response. Cell supernatants were assayed by ELISA (4 h for TNF-α and 24 h for MCP-1 after LPS challenge). Data are mean ± SEM, n = 4. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; Veh: vehicle (DMSO).

Article Snippet: All primer pairs were synthesized by Integrated DNA Technologies (Coralville, IA). siRNAs for MK2 (m) (CAT# sc-35856), TTP (m) (CAT# sc-36761), and scramble siRNA (CAT# sc-374305) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Incubation, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Pharmacological research

Article Title: Degradation of MK2 with natural compound andrographolide: A new modality for anti-inflammatory therapy.

doi: 10.1016/j.phrs.2023.106861

Figure Lengend Snippet: Fig. 3. The anti-inflammatory effects of AG are MK2-dependent and involve TTP. (A–B) Effects of AG on LPS-induced secretion levels of A. TNF-α and B. MCP-1 by NC and MK2-OE RAW264.7 cells. Cell supernatants were assayed by ELISA. Results were expressed as percentages of the LPS-treated control group. Data are mean ± SEM, n = 3. C. Schematic diagram illustrating the proposed mechanism of action of AG. D. The transfection efficiency of si-TTP. Cell lysates were assayed by immunoblotting. GAPDH was used as the loading control (n = 2). E. Effects of AG, PF-3644022, and p50-i on LPS-induced TNF-α production by NC and si-TTP- transfected RAW264.7 cells. Cells were pre-incubated with test compounds for 1 h and then treated with LPS (100 ng/mL) for 24 h. Cell supernatants were assayed by ELISA. Results were expressed as percentages of the LPS-treated NC group. Data are mean ± SEM, n = 3. F. Effects of AG on LPS-induced mRNA expression level of TNF-α in NC and si-TTP-transfected RAW264.7 cells. Cells were pre-incubated with AG (10 μM) for 1 h and then treated with LPS (100 ng/mL) for 4 h. Quantitative PCR was performed. The amount of mRNA was normalized to β-actin expression and was presented as fold of control. Data are mean ± SEM, n = 3. (G–H) Effects of AG on mRNA and protein levels of TTP in RAW264.7 cells. G. Cells were incubated with AG (20 μM) for the indicated times. Cell lysates were analyzed by immunoblotting. GAPDH was used as the loading control (n = 3). H. Cells were incubated with increasing concentrations of AG (i.e., 5, 10, 20, 50 μM) for 4 h and quantitative PCR was performed. The amount of mRNA was normalized to β-actin expression and was presented as fold of untreated control. Data are mean ± SEM, n = 4. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NC, negative control.

Article Snippet: All primer pairs were synthesized by Integrated DNA Technologies (Coralville, IA). siRNAs for MK2 (m) (CAT# sc-35856), TTP (m) (CAT# sc-36761), and scramble siRNA (CAT# sc-374305) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Transfection, Western Blot, Incubation, Expressing, Real-time Polymerase Chain Reaction, Negative Control

Journal: Pharmacological research

Article Title: Degradation of MK2 with natural compound andrographolide: A new modality for anti-inflammatory therapy.

doi: 10.1016/j.phrs.2023.106861

Figure Lengend Snippet: Fig. 5. AG promotes the ubiquitination and proteasomal degradation of MK2. A. Effects of AG on mRNA expression of MK2 in RAW264.7 cells. Cells were incubated with increasing concentrations of AG (i.e., 5, 10, 20, 50 μM) for 4 h and quantitative PCR was performed. The amount of mRNA was normalized to β-actin expression and was presented as fold of untreated control. Data are mean ± SEM, n = 4. B. Effects of AG on MK2 protein expression levels in the presence of MG132. Cells were pre-incubated with MG132 for 1 h and then treated with AG for another 2 h. Cell lysates were analyzed by immunoblotting. GAPDH was used as loading control. Band intensities were analyzed by ImageJ, and the MK2/GAPDH ratios relative to DMSO vehicle control (0.05 %) are indicated. Data are mean ± SEM, n = 3. C. Time-dependent effect of CHX and AG on MK2 protein levels. RAW264.7 cells were incubated with CHX (1 μg/mL) or AG (50 μM) for the indicated times. Cell lysates were analyzed by immunoblotting. Band intensities were analyzed by ImageJ, and the MK2/GAPDH ratios relative to t = 0 min are indicated. Data are mean, n = 3. D. Effects of AG on MK2 protein expression levels in the presence of SB203580. Cells were pre-incubated with SB203580 (10 μM) for 1 h and then treated with AG (50 μM) for another 2 h. Cell lysates were analyzed by immunoblotting. GAPDH was used as the loading control (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; Veh, vehicle (DMSO).

Article Snippet: All primer pairs were synthesized by Integrated DNA Technologies (Coralville, IA). siRNAs for MK2 (m) (CAT# sc-35856), TTP (m) (CAT# sc-36761), and scramble siRNA (CAT# sc-374305) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Ubiquitin Proteomics, Expressing, Incubation, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Pharmacological research

Article Title: Degradation of MK2 with natural compound andrographolide: A new modality for anti-inflammatory therapy.

doi: 10.1016/j.phrs.2023.106861

Figure Lengend Snippet: Fig. 6. AG binds to the activation loop of MK2 located at the interface between p38α and MK2. A. X-ray crystallography of p38α-MK2 complex obtained from PDB (ID: 2OZA). B. Docking of AG on MK2 (chain A). Blind docking (rigid residues) was performed using iGEMDOCK v.2.1. with 100 solutions. When the 100 conformers were sorted according to their energies, 90 % of the conformers with the lowest energies docked around a site adjacent to the activation loop. C. The binding site residues are colored. The figures were generated using the program Chimera [38]. D. CETSA for MK2. Total RAW264.7 cell lysates were treated with DMSO (0.05 %), AG (50 μM) and PF-3644022 (10 μM) for 1 h. Aliquots were subjected to gradient heat treatment and the soluble protein fraction was analyzed by immunoblotting. KHSRP was used as the loading control. Band intensities were analyzed by ImageJ and normalized to that at 37 ◦C to obtain the CETSA melt curves of MK2. Results were expressed as percentages of the unheated control group. Data are mean ± SEM, n = 3. E. Effects of AG on the phosphorylation of MK2 by constitutively-active p38α. AG was pre-incubated with recombinant MK2 10 min prior to the addition of p38α (4:1 molar ratio) and ATP (150 μM). The amount of phosphorylated MK2 was chased with phosphosite-specific antibodies that recognize Thr222 or Thr334 (n = 3). F. Kinase activity of MK2 measured by ADP-Glo kinase assay. Activated recombinant MK2 was pre-incubated with DMSO, AG, and PF3644022 in increasing concentrations for 10 min prior to the addition of HSP27 peptide substrate and ATP. The kinase reaction was allowed for 1 h before an ADP-Glo kinase assay. Results were expressed as percentages of the untreated control group. Data are mean ± SEM, n = 3.

Article Snippet: All primer pairs were synthesized by Integrated DNA Technologies (Coralville, IA). siRNAs for MK2 (m) (CAT# sc-35856), TTP (m) (CAT# sc-36761), and scramble siRNA (CAT# sc-374305) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Activation Assay, Binding Assay, Generated, Western Blot, Control, Phospho-proteomics, Incubation, Recombinant, Activity Assay, Kinase Assay

Journal: Pharmacological research

Article Title: Degradation of MK2 with natural compound andrographolide: A new modality for anti-inflammatory therapy.

doi: 10.1016/j.phrs.2023.106861

Figure Lengend Snippet: Fig. 8. Preliminary structure-activity relationship study on AG on MK2 degrader. A. Chemical structure of AG, and its derivatives AG1 and AG2. B. Effects of AG, AG1 and AG2 on MK2 protein levels. RAW264.7 cells were incubated with AG (20 μM), AG1 (50 μM), AG2 (50 μM) for 4 h. Cell lysates were analyzed by immunoblotting (n = 3). C. Effects of AG on LPS-induced protein levels of TNF-α. RAW264.7 cells were pre-incubated with AG (20 μM), AG1 (50 μM), AG2 (50 μM) for 1 h and then treated with LPS (100 ng/mL) for 4 h. Cell supernatants were assayed by ELISA. Data are mean ± SEM, n = 4.

Article Snippet: All primer pairs were synthesized by Integrated DNA Technologies (Coralville, IA). siRNAs for MK2 (m) (CAT# sc-35856), TTP (m) (CAT# sc-36761), and scramble siRNA (CAT# sc-374305) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Activity Assay, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of investigative dermatology

Article Title: MAPKAP kinase 2 (MK2)-dependent and independent models of blister formation in pemphigus vulgaris

doi: 10.1038/jid.2013.224

Figure Lengend Snippet: Activated MK2, demonstrated by immunohistochemical staining of pemphigus skin biopsy samples with an antibody specific for phospho-MK2, was markedly increased in PV (PV1-4) and PF (PF1-2) lesional skin keratinocytes. No significant activation was observed in normal human skin or PV non-lesional keratinocytes (arrows indicate focal activation in perilesional keratinocytes). Normal rabbit serum was used as antibody negative control. Activated MK2 is primarily observed in the cytoplasm of keratinocytes (PV4 and PF1, inset). Scale bar=100μm.

Article Snippet: Other reagents included p38 inhibitor (SB202190), hydrogen peroxide, and puromycin (Sigma-Aldrich, St. Louis, MO); MK2 inhibitor III, MK2 shRNA and control shRNA lentiviral particles (Santa Cruz Biotechnology).

Techniques: Immunohistochemical staining, Staining, Activation Assay, Negative Control

Journal: The Journal of investigative dermatology

Article Title: MAPKAP kinase 2 (MK2)-dependent and independent models of blister formation in pemphigus vulgaris

doi: 10.1038/jid.2013.224

Figure Lengend Snippet: A) Pathogenic (P) mAb activates MK2 in a dose-dependent manner (treatment 2 hours), while nonpathogenic (NP) mAb does not, similar to positive controls for p38 activation by P mAb and oxidative stress (H 2 O 2 ). B) Peak activation of MK2 by 50 μg/mL P mAb occurs at 2 hours. C) MK2 translocates from the nucleus to the cytosol after treatment of keratinocytes with P mAb. MK2 protein levels in the cytosolic and nuclear fractions (co-fractionation with beta-tubulin and histone, respectively) were detected by immunoblot. D) P mAb and H 2 O 2 , but not NP mAb, cause MK2 translocation from the nucleus to the cytosol at 4 hours, demonstrated by immunofluorescence. Scale bar=20μm.

Article Snippet: Other reagents included p38 inhibitor (SB202190), hydrogen peroxide, and puromycin (Sigma-Aldrich, St. Louis, MO); MK2 inhibitor III, MK2 shRNA and control shRNA lentiviral particles (Santa Cruz Biotechnology).

Techniques: Activation Assay, Fractionation, Western Blot, Translocation Assay, Immunofluorescence

Journal: The Journal of investigative dermatology

Article Title: MAPKAP kinase 2 (MK2)-dependent and independent models of blister formation in pemphigus vulgaris

doi: 10.1038/jid.2013.224

Figure Lengend Snippet: A) Pretreatment of PHEK with an MK2-specific inhibitor (MK2I) for 2 hours, then PV mAb for 2 hours, blocks phosphorylation of HSP27 but not p38 in a dose-dependent pattern. B) MK2I rescues the loss of desmosomal Dsg3 caused by P mAb. PHEK were treated with DMSO, 2μM SB202190 (p38 inhibitor) or 2.5 μg/mL MK2I for 2 hours, followed by 50 μg/ml PV mAbs (P or NP) for 6 hours. Triton X-100-insoluble fractions were immunoblotted with antibodies as indicated. Data are representative of three independent experiments. C) P but not NP mAb (as in B ) causes loss of cell surface Dsg3 (green), which is inhibited by SB202190 and MK2I. Scale bar=20μm.

Article Snippet: Other reagents included p38 inhibitor (SB202190), hydrogen peroxide, and puromycin (Sigma-Aldrich, St. Louis, MO); MK2 inhibitor III, MK2 shRNA and control shRNA lentiviral particles (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics

Journal: The Journal of investigative dermatology

Article Title: MAPKAP kinase 2 (MK2)-dependent and independent models of blister formation in pemphigus vulgaris

doi: 10.1038/jid.2013.224

Figure Lengend Snippet: A) HaCat cells that stably express MK2 shRNA (shRNA) show markedly reduced MK2 and p38 protein levels compared to cells expressing control (Ctl) shRNA. Levels of histone H3 are shown as a loading control. B) Immunofluorescence staining of HaCat cells expressing MK2 or control (Ctl) shRNA confirms knockdown of MK2 expression 72 hours after transduction. C) shRNA silencing of MK2 expression decreases loss of cell surface Dsg3 (shown in red) 16 hours after treatment with pathogenic PV mAb (P) and oxidative stress (H 2 O 2 ). Scale bar=20μm.

Article Snippet: Other reagents included p38 inhibitor (SB202190), hydrogen peroxide, and puromycin (Sigma-Aldrich, St. Louis, MO); MK2 inhibitor III, MK2 shRNA and control shRNA lentiviral particles (Santa Cruz Biotechnology).

Techniques: Stable Transfection, shRNA, Expressing, Control, Immunofluorescence, Staining, Knockdown, Transduction

Journal: The Journal of investigative dermatology

Article Title: MAPKAP kinase 2 (MK2)-dependent and independent models of blister formation in pemphigus vulgaris

doi: 10.1038/jid.2013.224

Figure Lengend Snippet: 25 μg of pathogenic (P), but not nonpathogenic (NP), mAb caused Nikolsky blisters (arrows) 6 hours after mAb injection then mechanical shear stress in MK2-knockout (KO) and wild-type (WT) littermate control mice (A) , and wild-type mice pretreated with DMSO or MK2I (D). (B, E) Histologic analysis demonstrates suprabasal acantholysis in mice injected with P but not NP mAb. The extent of histologic blistering was significantly different between P and NP mAb, but similar in WT and MK2 KO mice injected with P mAb (C) , as well as wild-type mice injected with P mAb after pretreatment with DMSO or MK2I (F) . Scale bar=100 μm. **p<0.01; *p<0.05; NS, non-significant

Article Snippet: Other reagents included p38 inhibitor (SB202190), hydrogen peroxide, and puromycin (Sigma-Aldrich, St. Louis, MO); MK2 inhibitor III, MK2 shRNA and control shRNA lentiviral particles (Santa Cruz Biotechnology).

Techniques: Injection, Shear, Knock-Out, Control

Journal: The Journal of investigative dermatology

Article Title: MAPKAP kinase 2 (MK2)-dependent and independent models of blister formation in pemphigus vulgaris

doi: 10.1038/jid.2013.224

Figure Lengend Snippet: A) 16 hours after pathogenic (P) mAb injection, spontaneous blisters occur in wild-type (WT) (arrows) but not MK2-knockout (KO) mice. B) Histology shows suprabasal acantholysis in WT mice. 11/15 KO mice did not blister (left). 4/15 KO mice with gross blisters demonstrated superficial, not suprabasal, acantholysis (right). C) Suprabasal histologic blistering was significantly decreased in KO versus WT mice. D) SB202190 or MK2I prevents spontaneous blistering by P mAb (arrow). E) P but not NP mAbs cause suprabasal acantholysis, inhibited by SB202190 or MK2I, with focal blistering in some sections. F) Histologic blistering was significantly decreased by SB202190 and MK2I. Scale bar=100 μm. **p<0.01; *p<0.05; NS, non-significant.

Article Snippet: Other reagents included p38 inhibitor (SB202190), hydrogen peroxide, and puromycin (Sigma-Aldrich, St. Louis, MO); MK2 inhibitor III, MK2 shRNA and control shRNA lentiviral particles (Santa Cruz Biotechnology).

Techniques: Injection, Knock-Out

Journal: Biochemical Journal

Article Title: Phosphorylation of cAMP-specific PDE4A5 (phosphodiesterase-4A5) by MK2 (MAPKAPK2) attenuates its activation through protein kinase A phosphorylation

doi: 10.1042/bj20101184

Figure Lengend Snippet: Figure 4 Phosphorylation by MK2 attenuates the action of stimulatory phosphorylation by PKA

Article Snippet: COS1 cells were transfected using either Polyfect® reagent (Qiagen) or DEAE-dextran (Sigma–Aldrich) as described previously [10,11]. siRNAs The PolyFect® method of mammalian cell transfection (Qiagen) was used for the transfection of COS1 cells with an MK2 siRNA nucleotide from Santa Cruz Biotechnology, as reported previously [12].

Techniques: Phospho-proteomics

Journal: Biochemical Journal

Article Title: Phosphorylation of cAMP-specific PDE4A5 (phosphodiesterase-4A5) by MK2 (MAPKAPK2) attenuates its activation through protein kinase A phosphorylation

doi: 10.1042/bj20101184

Figure Lengend Snippet: Figure 5 MK2 phosphorylation of PDE4A5 alters its ability to regulate intracellular cAMP concentrations

Article Snippet: COS1 cells were transfected using either Polyfect® reagent (Qiagen) or DEAE-dextran (Sigma–Aldrich) as described previously [10,11]. siRNAs The PolyFect® method of mammalian cell transfection (Qiagen) was used for the transfection of COS1 cells with an MK2 siRNA nucleotide from Santa Cruz Biotechnology, as reported previously [12].

Techniques: Phospho-proteomics

Journal: Biochemical Journal

Article Title: Phosphorylation of cAMP-specific PDE4A5 (phosphodiesterase-4A5) by MK2 (MAPKAPK2) attenuates its activation through protein kinase A phosphorylation

doi: 10.1042/bj20101184

Figure Lengend Snippet: Figure 6 MK2 phosphorylation of PDE4A5 attenuates its ability to be reversibly recruited into intracellular aggregates by chronic rolipram treatment

Article Snippet: COS1 cells were transfected using either Polyfect® reagent (Qiagen) or DEAE-dextran (Sigma–Aldrich) as described previously [10,11]. siRNAs The PolyFect® method of mammalian cell transfection (Qiagen) was used for the transfection of COS1 cells with an MK2 siRNA nucleotide from Santa Cruz Biotechnology, as reported previously [12].

Techniques: Phospho-proteomics

Journal: Biochemical Journal

Article Title: Phosphorylation of cAMP-specific PDE4A5 (phosphodiesterase-4A5) by MK2 (MAPKAPK2) attenuates its activation through protein kinase A phosphorylation

doi: 10.1042/bj20101184

Figure Lengend Snippet: Figure 7 MK2 phosphorylation of PDE4A5 and its ability to be sequestered by partner proteins

Article Snippet: COS1 cells were transfected using either Polyfect® reagent (Qiagen) or DEAE-dextran (Sigma–Aldrich) as described previously [10,11]. siRNAs The PolyFect® method of mammalian cell transfection (Qiagen) was used for the transfection of COS1 cells with an MK2 siRNA nucleotide from Santa Cruz Biotechnology, as reported previously [12].

Techniques: Phospho-proteomics