For protein extraction, cells were washed with ice-cold PBS and lysed for 20 min on ice with RIPA (RadioImmunoPrecipitationAssay) buffer (150 mM NaCl, 50 mM Tris pH7.4, 1 % NP40, 1 % sodium-deoxycholat, 1 mM EDTA, 1 mM Na3VO4, 25 mM NaF, 1 mM PMSF, 5 mM beta-glycerolphosphat, protease inhibitor cocktail tablets (Complete mini; Roche)). Total cell lysates were cleared by centriguation (10 min, 4 °C, 10,000 g). Protein concentration was measured with the Bradford ProteinAssay (BioRad). Western blots were performed as described in [50 (link)] with the following antibodies: anti-Fibronectin: Santa Cruz, sc-9068; anti-ZO1: Zymed Laboratories, 33–9100; anti-E-Cadherin: BD Transduction Laboratories, 610181; anti-Vimentin: Sigma, V2258; anti-Actin: Sigma, A2066; anti-phospho-AKT (Ser473): Cell Signaling, 9271; anti-total-AKT: Cell Signaling, 9272; anti-phospho-ERK1/2: Sigma, M8159; anti-total-ERK1/2:Sigma,M5670; anti-Pan-Ras: Calbiochem, OP38.
For immunoprecipitation, cells were lysed as described for Western blotting. Equal amounts of protein were incubated with anti-v-H-Ras antibody (Calbiochem, OP01) overnight at 4 °C. Then ProteinA/G plus beads (Santa Cruz) were added and samples were incubated for 1 h at 4 °C. Thereafter, immune complexes were collected by centrifugation, washed twice with ice cold RIPA buffer and subjected to SDS PAGE and Western blotting.
For immunoprecipitation, cells were lysed as described for Western blotting. Equal amounts of protein were incubated with anti-v-H-Ras antibody (Calbiochem, OP01) overnight at 4 °C. Then ProteinA/G plus beads (Santa Cruz) were added and samples were incubated for 1 h at 4 °C. Thereafter, immune complexes were collected by centrifugation, washed twice with ice cold RIPA buffer and subjected to SDS PAGE and Western blotting.
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