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mouse anti-serca2 iid8  (Millipore)


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    Structured Review

    Millipore mouse anti-serca2 iid8
    Mouse Anti Serca2 Iid8, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+anti-serca2+iid8/anti+serca2/pm37561594-145-0-7
    Average 90 stars, based on 1 article reviews
    mouse anti-serca2 iid8 - by Bioz Stars, 2026-09
    90/100 stars

    Images

    Related Articles

    Expressing:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Western Blot:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Transfection:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Binding Assay:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Luciferase:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Reporter Assay:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Construct:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Sequencing:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Inhibition:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Activity Assay:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Negative Control:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Plasmid Preparation:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Real-time Polymerase Chain Reaction:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).

    Fluorescence:

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease.
    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Article Title: HIF-1 regulation of miR-29c impairs SERCA2 expression and cardiac contractility
    Article Snippet: Antibodies used in this study were purchased from the following sources: rabbit anti-mouse HIF-1α (NB100-479, Novus Biologicals), mouse anti-rabbit GAPDH (clone GAPDH-71.1, Sigma), mouse anti-SERCA2 (clone IID8, Sigma), goat anti-human SERCA2 antibody (N-19, Santa Cruz Biotechnology), mouse anti-β-actin (clone AC-74, Sigma), mouse anti-human Akt (40D4, Cell Signaling), and rabbit anti-mouse phospho-Akt (Ser 473 , Cell Signaling). miRNA inhibitors (antimirs) were locked nucleic acid oligonucleotides purchased from Exiqon (now Qiagen).



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    Image Search Results


    ( A ) Immunoblot of SERCA2 in lysates from ATP2A2 wild-type (WT, +/+), heterozygous (HET, +/-), and homozygous knockout (KO, -/-) cells. hTERT-immortalized human epidermal keratinocytes (THEKs) were differentiated in E-medium for 72 hours before lysate harvesting; data represent 3 independent experiments; and β-actin is a loading control. ( B ) Immunofluorescence of SERCA2 (green) in WT, HET, and KO THEKs; images are representative of 14 independent high-powered fields (hpf) per genotype; Hoechst (blue) stains nuclei; scale bar = 10 μm. ( C ) Mechanical dissociation of monolayers from control (+/+), HET (+/-), and KO (-/-) cells grown in 1.3 mM CaCl 2 for 72 hours prior to using dispase to release intact monolayers; representative images of fragmented monolayers transferred into 6-well cell culture plates after mechanical stress are shown. ( D ) Graphs display mean ± SD of the number of epithelial fragments with data plotted for N = 3 biological replicates; P values from 1-way ANOVA with Dunnett’s adjustment for multiple comparisons. ( E ) Change (Δ) in intensity of GCaMP in control (+/+), HET (+/-), and KO (-/-) THEKs from baseline at t = 0 seconds with addition of 1 mM CaCl 2 at t = 60 seconds (arrow); data plotted as mean ± SEM from N = 3 independent experiments per genotype. ( F ) Representative fluorescence images of GCaMP in WT, HET, and KO cells in low CaCl 2 (left) or high CaCl 2 (right); scale bar = 10 μm.

    Journal: JCI Insight

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease

    doi: 10.1172/jci.insight.170739

    Figure Lengend Snippet: ( A ) Immunoblot of SERCA2 in lysates from ATP2A2 wild-type (WT, +/+), heterozygous (HET, +/-), and homozygous knockout (KO, -/-) cells. hTERT-immortalized human epidermal keratinocytes (THEKs) were differentiated in E-medium for 72 hours before lysate harvesting; data represent 3 independent experiments; and β-actin is a loading control. ( B ) Immunofluorescence of SERCA2 (green) in WT, HET, and KO THEKs; images are representative of 14 independent high-powered fields (hpf) per genotype; Hoechst (blue) stains nuclei; scale bar = 10 μm. ( C ) Mechanical dissociation of monolayers from control (+/+), HET (+/-), and KO (-/-) cells grown in 1.3 mM CaCl 2 for 72 hours prior to using dispase to release intact monolayers; representative images of fragmented monolayers transferred into 6-well cell culture plates after mechanical stress are shown. ( D ) Graphs display mean ± SD of the number of epithelial fragments with data plotted for N = 3 biological replicates; P values from 1-way ANOVA with Dunnett’s adjustment for multiple comparisons. ( E ) Change (Δ) in intensity of GCaMP in control (+/+), HET (+/-), and KO (-/-) THEKs from baseline at t = 0 seconds with addition of 1 mM CaCl 2 at t = 60 seconds (arrow); data plotted as mean ± SEM from N = 3 independent experiments per genotype. ( F ) Representative fluorescence images of GCaMP in WT, HET, and KO cells in low CaCl 2 (left) or high CaCl 2 (right); scale bar = 10 μm.

    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Techniques: Western Blot, Knock-Out, Immunofluorescence, Cell Culture, Fluorescence

    ( A ) Results from bulk RNA sequencing of SERCA2 HET versus control cells. Volcano plot depicts log 2 fold-changes of genes significantly downregulated (blue) and upregulated (red) with cutoff (dashed line) 0.05 for adjusted P value. ( B ) Gene ontology (GO) analysis of transcripts significantly altered (adjusted P ≤ 0.05) in HET cells revealed upregulation (red) in genes controlling ER stress and growth factor signaling and downregulation (blue) of genes controlling epidermal differentiation and antiviral response. ( C ) Control (+/+), HET (+/-), or KO (-/-) THEKs were differentiated in E-medium for 24 hours, and mRNA transcripts were quantified by reverse transcription quantitative PCR (RT-qPCR). Graphs display mean ± SD with plotted values from N = 4 biological replicates; P values from 1-way ANOVA with Dunnett’s adjustment for multiple comparisons. DSG1, desmoglein 1; KRT, keratin.

    Journal: JCI Insight

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease

    doi: 10.1172/jci.insight.170739

    Figure Lengend Snippet: ( A ) Results from bulk RNA sequencing of SERCA2 HET versus control cells. Volcano plot depicts log 2 fold-changes of genes significantly downregulated (blue) and upregulated (red) with cutoff (dashed line) 0.05 for adjusted P value. ( B ) Gene ontology (GO) analysis of transcripts significantly altered (adjusted P ≤ 0.05) in HET cells revealed upregulation (red) in genes controlling ER stress and growth factor signaling and downregulation (blue) of genes controlling epidermal differentiation and antiviral response. ( C ) Control (+/+), HET (+/-), or KO (-/-) THEKs were differentiated in E-medium for 24 hours, and mRNA transcripts were quantified by reverse transcription quantitative PCR (RT-qPCR). Graphs display mean ± SD with plotted values from N = 4 biological replicates; P values from 1-way ANOVA with Dunnett’s adjustment for multiple comparisons. DSG1, desmoglein 1; KRT, keratin.

    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Techniques: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    ( A ) Control or HET cells were differentiated in E-medium ± MEK inhibitors for 24 hours, and then transcripts were measured by RT-qPCR; graphs show mean ± SD from N = 4 replicates; P values from ANOVA (Brown-Forsythe) with Dunnett’s T3 adjustment for multiple comparisons. ( B ) Immunostaining KRT10 (and Hoechst) in control or HET cells treated with DMSO or 1 μM trametinib (Tram) for 48 hours; images represent 2 independent experiments using 2 control and 2 SERCA2-deficient lines; scale bar = 50 μm. ( C ) Quantification of KRT10 immunostaining in control versus SERCA2-deficient cells treated with DMSO or 1 μM Tram for 24 hours; data shown as a box plot of 25th–75th percentile with line at the median from N ≥ 21 nonoverlapping hpf per group; control mean normalized to 1; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons. ( D ) Immunoblot of KRT10 and KRT14 (β-actin loading control) in lysates from control versus HET cells treated with DMSO, 1 μM dabrafenib (Dab), or MEK inhibitors (1 μM Tram; 10 μM U0126; 10 μM PD184; 20 μM PD980); data represent 2 independent experiments. ( E ) Quantification of fragments of HET and KO monolayers treated with DMSO or 1 μM trametinib for 48 hours; graphs display mean ± SD for N = 3 replicates; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons. ( F ) Images of 6-well culture plates containing fragmented monolayers of control or HET cells treated with DMSO or 20 μM PD980 for 24 hours. ( G ) Quantification of fragments of control, HET, or KO monolayers treated with DMSO or 20 μM PD980 for 24 hours; graphs display mean ± SD for N = 3 replicates; P values from 1-way ANOVA with Dunnett’s adjustment for multiple comparisons.

    Journal: JCI Insight

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease

    doi: 10.1172/jci.insight.170739

    Figure Lengend Snippet: ( A ) Control or HET cells were differentiated in E-medium ± MEK inhibitors for 24 hours, and then transcripts were measured by RT-qPCR; graphs show mean ± SD from N = 4 replicates; P values from ANOVA (Brown-Forsythe) with Dunnett’s T3 adjustment for multiple comparisons. ( B ) Immunostaining KRT10 (and Hoechst) in control or HET cells treated with DMSO or 1 μM trametinib (Tram) for 48 hours; images represent 2 independent experiments using 2 control and 2 SERCA2-deficient lines; scale bar = 50 μm. ( C ) Quantification of KRT10 immunostaining in control versus SERCA2-deficient cells treated with DMSO or 1 μM Tram for 24 hours; data shown as a box plot of 25th–75th percentile with line at the median from N ≥ 21 nonoverlapping hpf per group; control mean normalized to 1; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons. ( D ) Immunoblot of KRT10 and KRT14 (β-actin loading control) in lysates from control versus HET cells treated with DMSO, 1 μM dabrafenib (Dab), or MEK inhibitors (1 μM Tram; 10 μM U0126; 10 μM PD184; 20 μM PD980); data represent 2 independent experiments. ( E ) Quantification of fragments of HET and KO monolayers treated with DMSO or 1 μM trametinib for 48 hours; graphs display mean ± SD for N = 3 replicates; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons. ( F ) Images of 6-well culture plates containing fragmented monolayers of control or HET cells treated with DMSO or 20 μM PD980 for 24 hours. ( G ) Quantification of fragments of control, HET, or KO monolayers treated with DMSO or 20 μM PD980 for 24 hours; graphs display mean ± SD for N = 3 replicates; P values from 1-way ANOVA with Dunnett’s adjustment for multiple comparisons.

    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Techniques: Quantitative RT-PCR, Immunostaining, Western Blot

    ( A ) Immunostaining of KRT10 (and Hoechst) in THEKs treated with DMSO, 1 μM TG, or 1 μM TG plus 1 μM Tram (TG + Tram) for 48 hours; scale bar = 50 μm. ( B ) Quantification of KRT10 immunostaining in THEKs treated with DMSO, TG, or TG plus Tram; data shown as a box plot of the 25th–75th percentile with a line at the median from N ≥ 31 nonoverlapping hpf from 2 independent THEK lines for both control and SERCA2-deficient cells; control mean normalized to 1; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons. ( C ) Representative images of fragmented monolayers transferred into 6-well cell culture plates are shown for NHEKs treated with DMSO versus 1 μM TG alone or with 1 μM dabrafenib versus an MEK inhibitor (1 μM Tram, 10 μM U0126, or 20 μM PD980) for 24 hours. ( D ) Quantification of epithelial fragments of NHEK monolayers; bar graphs display mean ± SD with data points for N = 6 biological replicates; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons. ( E ) H&E-stained tissue cross sections of organotypic cultures of NHEKs treated with DMSO, 1 μM TG, or 1 μM TG plus 20 μM PD980, the latter displaying improved keratinocyte cohesion and normalization of cornification; scale bar = 100 μm. ( F ) Quantification of retained nuclei in cornified layers of organotypic cultures treated with the indicated inhibitors for 48 hours; graph displays mean ± SD with plotted values averaged from ≥49 nonoverlapping hpf per condition from N = 4 biological replicates; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons.

    Journal: JCI Insight

    Article Title: Targeting SERCA2 in organotypic epidermis reveals MEK inhibition as a therapeutic strategy for Darier disease

    doi: 10.1172/jci.insight.170739

    Figure Lengend Snippet: ( A ) Immunostaining of KRT10 (and Hoechst) in THEKs treated with DMSO, 1 μM TG, or 1 μM TG plus 1 μM Tram (TG + Tram) for 48 hours; scale bar = 50 μm. ( B ) Quantification of KRT10 immunostaining in THEKs treated with DMSO, TG, or TG plus Tram; data shown as a box plot of the 25th–75th percentile with a line at the median from N ≥ 31 nonoverlapping hpf from 2 independent THEK lines for both control and SERCA2-deficient cells; control mean normalized to 1; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons. ( C ) Representative images of fragmented monolayers transferred into 6-well cell culture plates are shown for NHEKs treated with DMSO versus 1 μM TG alone or with 1 μM dabrafenib versus an MEK inhibitor (1 μM Tram, 10 μM U0126, or 20 μM PD980) for 24 hours. ( D ) Quantification of epithelial fragments of NHEK monolayers; bar graphs display mean ± SD with data points for N = 6 biological replicates; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons. ( E ) H&E-stained tissue cross sections of organotypic cultures of NHEKs treated with DMSO, 1 μM TG, or 1 μM TG plus 20 μM PD980, the latter displaying improved keratinocyte cohesion and normalization of cornification; scale bar = 100 μm. ( F ) Quantification of retained nuclei in cornified layers of organotypic cultures treated with the indicated inhibitors for 48 hours; graph displays mean ± SD with plotted values averaged from ≥49 nonoverlapping hpf per condition from N = 4 biological replicates; P values from 1-way ANOVA with Tukey’s adjustment for multiple comparisons.

    Article Snippet: Mouse anti-SERCA2 (IID8; catalog MAB2636) is from MilliporeSigma.

    Techniques: Immunostaining, Cell Culture, Staining

    (A) 293T cells were transfected with a plasmid containing kGPCR. kGPCR was purified with anti-Flag affinity chromatography. kGPCR-interacting proteins were analyzed by commassie staining and photographed, identified by mass spectrometry analysis. A separating line indicates a lane that was removed between lanes of vector and kGPCR. Note, heat-denatured kGPCR oligomerized and migrated slowly. (B) 293T cells were transfected with plasmids containing SERCA2 or kGPCR. Centrifuged cell extract was precipitated with anti-HA (kGPCR). Precipitated proteins and whole cell lysates were analyzed by immunoblot with indicated antibodies. (C and D) HUVEC endothelial stable cells were analyzed by proximity ligation assay (red) and stained with DAPI. Cells were analyzed by confocal microscopy (C). The intensity of proximity ligation was semi-quantitatively determined by counting more than 100 cells (D). (E) 293T cells were transfected with a plasmid containing Flag-tagged SERCA2 without or with a plasmid containing kGPCR. SERCA2 was precipitated with anti-Flag agarose and analyzed by ATPase activity in vitro. Precipitated SERCA2 and whole cell lysates were analyzed by immunoblot (insert). (F) 293T cells were transfected with an NFAT reporter cocktail, a plasmid containing kGPCR and increasing amount of a plasmid containing SERCA2. NFAT activation was determined by luciferase reporter assays.

    Journal: PLoS Pathogens

    Article Title: Herpesviral G Protein-Coupled Receptors Activate NFAT to Induce Tumor Formation via Inhibiting the SERCA Calcium ATPase

    doi: 10.1371/journal.ppat.1004768

    Figure Lengend Snippet: (A) 293T cells were transfected with a plasmid containing kGPCR. kGPCR was purified with anti-Flag affinity chromatography. kGPCR-interacting proteins were analyzed by commassie staining and photographed, identified by mass spectrometry analysis. A separating line indicates a lane that was removed between lanes of vector and kGPCR. Note, heat-denatured kGPCR oligomerized and migrated slowly. (B) 293T cells were transfected with plasmids containing SERCA2 or kGPCR. Centrifuged cell extract was precipitated with anti-HA (kGPCR). Precipitated proteins and whole cell lysates were analyzed by immunoblot with indicated antibodies. (C and D) HUVEC endothelial stable cells were analyzed by proximity ligation assay (red) and stained with DAPI. Cells were analyzed by confocal microscopy (C). The intensity of proximity ligation was semi-quantitatively determined by counting more than 100 cells (D). (E) 293T cells were transfected with a plasmid containing Flag-tagged SERCA2 without or with a plasmid containing kGPCR. SERCA2 was precipitated with anti-Flag agarose and analyzed by ATPase activity in vitro. Precipitated SERCA2 and whole cell lysates were analyzed by immunoblot (insert). (F) 293T cells were transfected with an NFAT reporter cocktail, a plasmid containing kGPCR and increasing amount of a plasmid containing SERCA2. NFAT activation was determined by luciferase reporter assays.

    Article Snippet: Commercial antibodies used in this study include mouse anti-HA monoclonal antibody and agarose (Sigma), mouse anti- β-Actin monoclonal antibody (Abcam), rabbit anti-COX-2 polyclonal antibody (Abcam), rabbit anti-RCAN1 polyclonal antibody (Sigma), mouse anti-SERCA2 (IID8) monoclonal antibody (Santa cruz), mouse anti-calcineurin Aα (Santa cruz).

    Techniques: Transfection, Plasmid Preparation, Purification, Affinity Chromatography, Staining, Mass Spectrometry, Western Blot, Proximity Ligation Assay, Confocal Microscopy, Ligation, Activity Assay, In Vitro, Activation Assay, Luciferase