|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 5. Association with lipid rafts of human CNTF-R, human gp130, and canine LIF-R in stably transfected and parental MDCK cells. MDCK-CNTF-R (A-C), MDCK-gp130 (D-F), parental MDCK (G-I) and MDCK-CNTF-R (K-M) cells were grown on tissue culture dishes and lysed at 4°C using three different protocols: a detergent-free extraction with sodium carbonate buffer, pH 11 (A,D,G,K), extraction with 1% Triton X-100 (B,E,H,L), and extraction with 1% Brij 58 (C,F,I,M) (see Materials and Methods for details). Lysates were subsequently homogenised and adjusted to 45% (A,D,G,K) or 40% sucrose (B,C,E,F,H,I,L,M) respectively. Subcellular fractions were obtained by ultracentrifugation at 4°C in a sucrose gradient (45/35/5% or 40/30/5% sucrose) for 16-20 hours at 192,000 g in a Beckman SW40 rotor. 1 ml fractions were collected, TCA-precipitated and proteins were analysed by 10% SDS-PAGE. Proteins were transferred to a PVDF membrane and the CNTF-R (A-C), gp130 (D-F), or the LIF-R (G-I) and also the endogenous caveolin-1 (A-I) were sequentially detected with specific antibodies and HRP-conjugated secondary antibodies. In the control panels (K-M) blots were incubated with specific antibodies to Rab5 or caveolin-1 and respective HRP-conjugated secondary antibodies. The proteins were visualised using the ECL+plus system.
|
