Journal: Nature Communications
Article Title: RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma
Figure Lengend Snippet: Compensatory functions of BRAF and CRAF for cell proliferation and tumour growth in NRAS Q61K -induced murine melanoma. ( a , e ) A melanoma from an untreated Braf f/f ;Craf +/+ ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o or Braf +/+ ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o mouse ( a , e , respectively) was cut into small pieces and subcutaneously grafted into two groups of nude mice and experimented as in Fig. 2a . These experiments required 48 Swiss Nu/Nu females (6-week-old) for each primary tumour from a 5-month-old female and a 1-year-old male on a SV129/C57Bl6 mixed genetic background, respectively. ( b , f ) Western blot analysis for BRAF and CRAF expression at the end of treatment by tamoxifen or vehicle in three individual and representative tumours from a , e , respectively. β-actin is used as a loading control. ( c , g ) Growth curve analysis of melanoma cell culture established from an untreated Braf f/f ;Craf +/+ ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o or Braf +/+ ;Craf f/f ;Tyr::NRAS Q61K / o ;Ink4a +/− ;Tyr::CreERT2 / o primary mouse tumour ( c , g , respectively) in response to 4OHT or DMSO for 9 days as in Fig. 2c . ( d , h ) Western blot analysis of BRAF and CRAF protein levels and MEK and ERK activation levels in protein lysates from culture in c , g respectively, as in Fig. 2b . All data are represented as mean±s.d.
Article Snippet: Membranes were then probed overnight at 4 °C with the appropriated primary antibodies: anti-ARAF (sc408, Santa Cruz, 1:500), anti-BRAF (sc5284, Santa Cruz, 1:2,000), anti-CRAF (#610151, BD Biosciences, 1:2,000), anti-NRAS (sc519, Santa Cruz, 1:2,000), anti-pMEK (#9121, Cell Signaling, 1:1,000), anti-MEK (sc219, Santa Cruz, 1:2,000), anti-pERK (M8159, Sigma, 1:2,000), anti-ERK (sc93, Santa Cruz, 1:2,000) and anti-βactin (A1978, Sigma, 1:5,000) antibodies.
Techniques: Mouse Assay, Western Blot, Expressing, Cell Culture, Activation Assay