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Fig. 6. Effect of receptor activation on lipid raft association. MDCK-CNTF-R cells (A), MDCK-gp130 (B,C), and parental MDCK cells (D) were stimulated with CNTF (A), IL-6/sIL-6R (B) or LIF (C+D) for 30 minutes at 37°C and lysed at 4°C using the Triton X-100 (A) or the Brij 58 (B-D) protocol (see Materials and Methods). After ultracentrifugation in a 40/30/5% sucrose gradient at 4°C for 16-20 hours at 192,000 g in a SW40 rotor 1 ml fractions were collected, trichloracetic acid-precipitated, and proteins analysed by 10% SDS-PAGE and western blotting. Human CNTF-R (A), human gp130 (B,C), and the endogenous LIF-R (D), respectively, as well as endogenous caveolin-1 (A-D) were sequentially detected with specific antibodies and HRP-conjugated secondary antibodies. The proteins were visualised using the ECL+plus system.
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