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. 2010 Jan 22;140(2):209–221. doi: 10.1016/j.cell.2009.12.040

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© 2010 ELL & Excerpta Medica.

Open Access under CC BY 3.0 license

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Tumors Induced by Kinase-Dead Braf and Oncogenic Kras Are Melanoma

(A) Photomicrograph of a section of tumor subjected to immunohistochemical analysis with antibodies against S100.

(B) RT-PCR analysis revealing expression of tyrosinase (Tyr), Dct, Pax3 and silver/gp100 (Si) in two independent tumors and kidney (control). GAPDH is used as a loading control.

(C) PCR-mediated genotyping for wild-type Braf (Braf^WT), Braf^+/LSL-D594A and Tyr::CreERT2^+/o alleles from a tumor sample, cells derived from the tumor and from kidney as a control.

(D) PCR amplified fragment for Kras from kidney and tumor samples. Shown below is the sequencing trace for codons 11–13, together with the DNA and protein sequence (single amino acid code).

(E) Endogenous CRAF was immunoprecipitated (IP) from cells derived from a tumor from a ^G12DKras/^D594ABraf mouse (Kras^+/LSL-G12D;Braf^+/LSL-D594A;Tyr::CreERT2^+/o), or from a tumor from a ^G12VKras mouse (β-actin^{+/LSL-G12VKras};Tyr::CreERT2^+/o). The immunocomplexes were western blotted (WB) for Braf and Craf, and the levels of Braf and Craf in the cell lysates are also shown.