therascreen kras pyro kit  (Qiagen)

Journal: PLoS ONE

Article Title: Expression of Abelson Interactor 1 (Abi1) Correlates with Inflammation, KRAS Mutation and Adenomatous Change during Colonic Carcinogenesis

doi: 10.1371/journal.pone.0040671

Figure Lengend Snippet: , Distribution of Abi1 expression in healthy and inflamed mucosa, hyperplastic polyps (HPP), sessile serrated polyps/adenomas (SSA/P), traditional serrated adenomas (TSA), tubular adenomas (TbA), invasive colorectal carcinoma (Ca) and metastases (Met). All values except BRAF-mutated TbA and carcinoma (each n = 1) are shown in box and whisker plot. Green squares represent maximum outliers, red squares represent minimum outliers. For inflamed mucosa, median, 1 st and 3 rd quartile are equal (score = 4). B , Statistical differences in Abi1 expression among all examined tissue specimens with respect to mutation status and, where applicable, microsatellite stability of each lesion. The lane for KRAS-mutated HPP is highlighted with a yellow background, the lane for KRAS-mutated invasive carcinoma is highlighted with a red background. The undermost line shows the number of examined samples in each group. M: healthy mucosa; IM: inflamed mucosa; HP wt, HP K, HP B: wild-type, KRAS-mutated and BRAF-mutated hyperplastic polyps; SP wt, SP K, SP B: wild-type, KRAS-mutated and BRAF-mutated sessile serrated polyps/adenomas; TA wt, TA K: wild-type and KRAS-mutated traditional serrated adenomas; TbA wt, TbA K: wild-type and KRAS-mutated tubular adenomas; CA wt, CA K, CA MI: wild-type, KRAS-mutated and microsatellite-instable carcinomas; Met wt, Met K: wild-type and KRAS-mutated metastases; n.s.: not significant; *p<0.1;** p<0.05;*** p<0.01.

Article Snippet: Pyrosequencing was then performed on the PyroMark Q24 System using the therascreen KRAS and BRAF Pyro Kits (all Qiagen, Hilden, Germany) according to the manfacturer’s protocol.

Techniques: Expressing, Whisker Assay, Mutagenesis

Journal: PLoS ONE

Article Title: Expression of Abelson Interactor 1 (Abi1) Correlates with Inflammation, KRAS Mutation and Adenomatous Change during Colonic Carcinogenesis

doi: 10.1371/journal.pone.0040671

Figure Lengend Snippet: , Abi1 immunoblotting of colorectal carcinoma whole cell line lysates with different KRAS/BRAF mutation status shows upregulation of Abi1 in KRAS-mutated SW620 and SW1116, but only a faint signal in BRAF-mutated Colo205 cells. B , Immunoblotting of CHD-1 and HDC-9 cell lysates show overexpression of Abi1 in CHD1 cells, while both cell lines express comparable amounts of PI3K (I). There is slightly stronger Akt phosphorylation in CHD1 compared to HDC9 cells. Application of 50 nM Wortmannin (WO) almost completely repressed the pAkt and Abi1 signals. Immunofluorescence microscopy shows strong cytoplasmatic and nuclear Abi1 staining in CHD-1 cells, but only a faint cytoplasmatic signal in HDC-9 cells (II). C, KRAS/BRAF mutation testing reveals an activating KRAS G13D mutation in CHD-1 (left lane), while HDC-9 cells are KRAS wild-type (central lane). Transfection of HDC-9 cells with a KRAS G12D-construct leads to appearance of a band indicating a KRAS G12D-mutation (right lane). Both cell lines are BRAF wild-type. D, Immunoblotting of HDC-9 after transfection, TNFalpha and Wortmannin treatment shows an increase in pErk1/2 and pAkt and overexpression of Abi1 upon transfection with constitutively active KRAS G12D (2 nd lane) compared to both control (3 rd lane) and to transfection with wild-type KRAS (1 st lane). Stimulation with TNFalpha also enhances phosphorylation of signaling proteins and leads to upregulation of Abi1 (4 th lane). The overexpression of Abi1 could be reversed by application of 50 nM Wortmannin (5 th lane).

Article Snippet: Pyrosequencing was then performed on the PyroMark Q24 System using the therascreen KRAS and BRAF Pyro Kits (all Qiagen, Hilden, Germany) according to the manfacturer’s protocol.

Techniques: Western Blot, Mutagenesis, Over Expression, Immunofluorescence, Microscopy, Staining, Transfection, Construct

Journal: PLoS ONE

Article Title: Expression of Abelson Interactor 1 (Abi1) Correlates with Inflammation, KRAS Mutation and Adenomatous Change during Colonic Carcinogenesis

doi: 10.1371/journal.pone.0040671

Figure Lengend Snippet: Ligand-binding to membrane-associated receptor tyrosine kinase (RTK) leads to an activation of RAS, which is constitutively activated in mutant KRAS (*). Activated KRAS activates - among others - both the B1 Rapidly accelerated fibrosarcoma (BRAF)- and Phosphatidylinositol-3-kinase (PI3K)-pathway, only the latter leading to activation of the Abi1/Sos1/Eps8 complex. Via activation of the small GTPase Rac, this leads to reorganization of the actin cytoskeleton and to a change in cellular shape. RTK: receptor tyrosine kinase; KRAS: Kirsten rat sarcoma; BRAF: B1 Rapidly accelerated fibrosarcoma ; PI3K: Phosphatidylinositol-3-kinase; Abi1: Abelson interactor 1; Eps8: Epidermal growth factor receptor kinase substrate ; Sos1: Son of sevenless homolog 1 ; Rac: Ras-related C3 botulinum toxin substrate.

Article Snippet: Pyrosequencing was then performed on the PyroMark Q24 System using the therascreen KRAS and BRAF Pyro Kits (all Qiagen, Hilden, Germany) according to the manfacturer’s protocol.

Techniques: Ligand Binding Assay, Activation Assay, Mutagenesis