mk2 shrna lentiviral particle  (Santa Cruz Biotechnology)

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: 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