图片
网站标志
点评详情
发布于:2018-11-9 19:41:52  访问:44 次 回复: 篇
版主管理 | 推荐 | 删除 | 删除并扣分
Cancer cell DNA [14]. Genomic amplification was defined as a greater than
The membranes have been analyzed by immunoblot applying the following antibodies, as indicated: mouse monoclonal anti-KRAS, -NRAS, and -HRAS PF-573228 FAK antibodies (sc-30, sc-31, and sc-29, respectively, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-actin antibody (Millipore); rabbit polyclonal anti-p44/42 MAP kinase, -phosho-p44/42 MAP kinase (Thr202/Tyr204), -Akt and -phospho-Akt (Ser473) antisera (Cell Signaling Technologies, Danvers, MA, USA).GTP-RAS pull-down assay The activation of RAS was detected applying an EZ-Detect Ras Activation Kit (Pierce, Rockford, IL, USA). Relative mRNA levels have been calculated by the comparative CT process employing GAPDH as an endogenous manage. The primer/probe sets utilised are shown in Additional file five. Fluorescence in situ hybridization (FISH) BACs that contained PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27486068 the KRAS locus (RP11-636P12) and chromosome 12q24.2 (RP11-91M21) have been labeled with Cy3 and Cy5, respectively, then incubated with slides ready with interphase and metaphase chromosomes. Nuclei have been counter-stained with 4‘,6-diamino-2-phenylindole (DAPI), and slides were analyzed working with a fluorescence microscope (Leica CW-4000). Mutational analysis of KRAS and PIK3CA Amplified genomic fragments were either sequenced straight, or subcloned working with the TOPO TA-cloning kit (Invitrogen) and then sequenced. No less than ten clones from two independent PCR assays per locus have been sequenced using M13 Forward and Reverse primers (Invitrogen). PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28893839 The sequences from the primers made use of for amplification of KRAS (exons 1 and two) and PIK3CA (exons 9 and 20) are shown in Added file 6. Immunoblot evaluation Cells were lysed in Lysis buffer containing 20 mM TrisHCl (pH7.five) buffer, 150 mM NaCl, 1 mM EDTA, 1 Tri-Page three of(page quantity not for citation purposes)BMC Cancer 2009, 9:http://www.biomedcentral.com/1471-2407/9/ton X, ten glycerol, ten mM NaF, 1 mM sodium vanadate, 50 mM -glycerophosphate, 1 mM phenylmethansulfonyl fluoride, 1 mM dithiothreitol, and a protease inhibitor cocktail (Roche, Mannheim, Germany). Proteins have been separated by SDS-PAGE and electroblotted onto an Immobilon-P membrane (Millipore, Billerica, MA, USA). The membranes have been analyzed by immunoblot working with the following antibodies, as indicated: mouse monoclonal anti-KRAS, -NRAS, and -HRAS antibodies (sc-30, sc-31, and sc-29, respectively, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-actin antibody (Millipore); rabbit polyclonal anti-p44/42 MAP kinase, -phosho-p44/42 MAP kinase (Thr202/Tyr204), -Akt and -phospho-Akt (Ser473) antisera (Cell Signaling Technology, Danvers, MA, USA).GTP-RAS pull-down assay The activation of RAS was detected using an EZ-Detect Ras Activation Kit (Pierce, Rockford, IL, USA). Briefly, cell lysate (500 g) was incubated with immobilized Raf1 Rasbinding domain fused to glutathione S-transferase (GSTRaf1-RBD). Precipitates had been washed three occasions, and bound proteins had been eluted by boiling for 5 minutes (min). Proteins had been resolved on a 12 polyacrylamide gel, transferred to an Immobilon-P membrane, and subjected to immunoblot evaluation applying anti-KRAS, -NRAS, or -HRAS antibodies. RNA interference A custom-designed KRAS siRNA (5‘-AGAGUGCCUUGACGAUACAdTdT-3‘), targeting a region of KRAS which is not connected with identified oncogenic mutations, was synthesized by Dharmacon (Lafayette, Co, USA).
共篇回复 每页10篇 页次:1/1
共篇回复 每页10篇 页次:1/1
我要回复
回复内容
验 证 码
看不清?更换一张
匿名发表 
脚注信息

家具制造企业网站 Copyright(C)2009-2010