antibodies against ccnd1 clone sp4  (Thermo Fisher)

Journal: Nature Communications

Article Title: Combined activation of MAP kinase pathway and β-catenin signaling cause deep penetrating nevi

doi: 10.1038/s41467-017-00758-3

Figure Lengend Snippet: Combined MAPK and β-catenin pathway activating mutations define deep penetrating nevi a β-catenin pathway mutations, affecting CTNNB1 or APC, and MAPK pathway mutations co-occur in deep penetrating nevi (DPN). Nevi with overlapping features of DPN and blue nevus harbor GNAQ activating mutations and are genetically distinct. BRAF activating mutations are mutually exclusive with MAP2K1 alterations. b CTNNB1 missense mutations affect codons of a critical domain that is phosphorylated and regulates subsequent ubiquitin-mediated degradation. c Indels of MAP2K1 cluster near a highly conserved lysine within the kinase catalytic domain. One small deletion affects the negative regulatory region (NRR). DD, docking domain for ERK1/2; NES, nuclear export signal; AL, activation loop within the kinase catalytic domain; PRD, proline-rich domain within the kinase catalytic domain; DVD, domain of versatile docking. d . MAP2K1 mutations activate MAP kinase signaling, which could be inhibited by the MEK inhibitor trametinib

Article Snippet: Immunohistochemical analysis was performed on archival FFPE tumor specimens using the following antibodies: CTNNB1 clone B-catenin 1 (Dako catalog# M3539 or IR702), CCND1 clone EP-12 (Dako, catalog #IR083) or clone SP4 (Thermo Scientific, catalog #RM-9104-R7).

Techniques: Ubiquitin Proteomics, Activation Assay

Journal: Nature Communications

Article Title: Combined activation of MAP kinase pathway and β-catenin signaling cause deep penetrating nevi

doi: 10.1038/s41467-017-00758-3

Figure Lengend Snippet: Mutant CTNNB1 confers increased cell volume, pigmentation and cyclin D1 levels in vitro. a Stably transduced melanocytes (melan-a) with BRAF V600E and CTNNB1 S33F demonstrate increased β-catenin activity as measured by TOP flash luciferase expression ( left ; P = 0.03), cell volume ( center ; P = 0.04) and melanin content ( right ; P = 0.007) as compared to melanocytes transduced with BRAF V600E alone. Mean with error bars showing s.e.m., unpaired Student’s t -test, three biological replicates performed. b Representative western blot analysis of melanocytes stably transduced with BRAF V600E and CTNNB1 S33F demonstrate increased cyclin D1 levels as compared to those tranduced with GFP or BRAF V600E . Data shown are representative of at least three independent experiments with similar results

Article Snippet: Immunohistochemical analysis was performed on archival FFPE tumor specimens using the following antibodies: CTNNB1 clone B-catenin 1 (Dako catalog# M3539 or IR702), CCND1 clone EP-12 (Dako, catalog #IR083) or clone SP4 (Thermo Scientific, catalog #RM-9104-R7).

Techniques: Mutagenesis, In Vitro, Stable Transfection, Activity Assay, Luciferase, Expressing, Transduction, Western Blot

Journal: Nature Communications

Article Title: Combined activation of MAP kinase pathway and β-catenin signaling cause deep penetrating nevi

doi: 10.1038/s41467-017-00758-3

Figure Lengend Snippet: Model of step-wise progression in DPN-like melanoma. BRAF mutation leads to a common nevus. Subsequent CTNNB1 mutation results in the phenotypic switch to DPN. Additional genetic alterations result full transformation to DPN-like melanoma

Article Snippet: Immunohistochemical analysis was performed on archival FFPE tumor specimens using the following antibodies: CTNNB1 clone B-catenin 1 (Dako catalog# M3539 or IR702), CCND1 clone EP-12 (Dako, catalog #IR083) or clone SP4 (Thermo Scientific, catalog #RM-9104-R7).

Techniques: Mutagenesis, Transformation Assay

Journal: Oncogene

Article Title: WIP1 modulates responsiveness to Sonic Hedgehog signaling in neuronal precursor cells and medulloblastoma

doi: 10.1038/onc.2016.96

Figure Lengend Snippet: ( A ) Schematic of construct used to generate ND2 : WIP1 transgenic mice, showing a 1-kb portion of the Neurod2 ( ND2 ) promoter (green arrow) driving expression of WIP1 (orange arrow; 1.8-kb). ( B ) DNA gel showing RT-PCR results from tail snips of ND2 : WIP1 transgenic founder mice using primers spanning exons 4 and 5 of WIP1 : expected 2485 base pair (bp) PCR product from endogenous Wip1 ; 180bp PCR product from WIP1 cDNA from expression construct in ( A ). C, empty template control; 1–15, founder mice; D1, PCR product using ND2 : WIP1 construct as template. DNA ladders shown on either side of gel, with notations for 200–2000bp. ( C ) WIP1 expression by real-time RT-PCR in the cerebellum of P7 ND2 : WIP1 transgenic, relative to Gapdh , compared to expression in the cerebellum of P7 wild-type (WT) C57Bl/6 mice, * p <0.005. ( D ) Hematoxylin and eosin stained P5 cerebella of WT and ND2 : WIP1 transgenic mice (left-hand panel). Dotted line, cerebellar lobules (I–X). Quantitation of cerebellar lobules in P5 WT and ND2 : WIP1 transgenic mice (right-hand panel) (n=3 cerebella per genotype), * p <0.05. ( E ) Immunohistochemical staining (left-hand panels) and quantitation (right-hand panels) of Ki-67 and the downstream marker of hedgehog pathway activation, Cyclin D1 (Ccnd1), in the external granule layer (EGL) of lobule X of the cerebellum of P5 wild-type, ND2 : WIP1 , and SmoA1 / SmoA1 mice, * p <0.05. Bar, EGL width. ( F ) Immunofluorescent staining (left-hand panels) and quantitation (right-hand panels) of Ki-67 and p27 Kip1 (p27), in the external granule layer (EGL) of the cerebellum of P5 wild-type, ND2 : WIP1 , and SmoA1 / SmoA1 mice (n=3 cerebella per genotype), * p <0.005. Ki67, red; p27, green; DAPI, blue. Bar, EGL width. ML, molecular layer; IGL, internal granule layer; HPF, high-power field. Error bars, standard deviation (SD) among replicates of at least three per group. Scale bars, 100µm. All experiments were repeated at least three times or in at least three distinct cerebella.

Article Snippet: Following antigen retrieval, tissues were blocked and incubated with α-Ki67 (1:500; Vector, VP-RMO4) or Ccnd1 (1:100; Thermo Scientific, SP4) overnight at 4°C, then incubated with biotinylated goat anti-rabbit IgG (1:300; Vector, BA-1000).

Techniques: Construct, Transgenic Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Quantitative RT-PCR, Staining, Quantitation Assay, Immunohistochemical staining, Marker, Activation Assay, Standard Deviation

Journal: Oncogene

Article Title: WIP1 modulates responsiveness to Sonic Hedgehog signaling in neuronal precursor cells and medulloblastoma

doi: 10.1038/onc.2016.96

Figure Lengend Snippet: ( A ) Immunohistochemical staining (left-hand panels) and quantitation (right-hand panels) of Ki-67+ and Cyclin D1 (Ccnd1)+ cells in the external granule layer of the cerebellum of P5 Wip1 +/+ and Wip1 −/− mice (n=3 cerebella per genotype), * p <0.005. Scale bar, 100µm. ( B ) Kaplan-Meier curves showing overall, MB-related survival of SmoA1 / SmoA1 ; Wip1 +/+, SmoA1 / SmoA1 ; Wip1 +/−, and SmoA1 / SmoA1 ; Wip1 −/− mice. ( C ) Kaplan-Meier curves showing overall, MB-related survival of Math1 -cre ER ; Ptc1 fl/fl; Wip1 +/+, Math1 -cre ER ; Ptc1 fl/fl; Wip1 +/−, and Math1 -cre ER ; Ptc1 fl/fl; Wip1 −/− mice following treatment with 1mg tamoxifen by oral gavage per mouse at P7.

Article Snippet: Following antigen retrieval, tissues were blocked and incubated with α-Ki67 (1:500; Vector, VP-RMO4) or Ccnd1 (1:100; Thermo Scientific, SP4) overnight at 4°C, then incubated with biotinylated goat anti-rabbit IgG (1:300; Vector, BA-1000).

Techniques: Immunohistochemical staining, Staining, Quantitation Assay