|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 8. Activation of Lyn induced by clustering of GM1. (A, left panel) PC12 cells treated with 5 µg/ml of laminin-1 for the indicated times in the presence of 100 ng/ml of NGF. Lyn was immunoprecipitated and subjected to immunoblotting using antibody against phosphorylated (Y396) Lyn (pLyn). The same blot was reprobed with anti-Lyn antibody. (Right panel) PC12 cells treated with 100 ng/ml of NGF and/or 5 µg/ml of laminin-1. Cells were lysed in 1% Triton and were fractionated on discontinuous sucrose gradients. Raft (R) and non-raft (NR) fractions were collected and subjected to immunoblotting using the indicated antibodies. Flotillin-1 was detected in the raft fraction. (B) PC12 cells were treated with 2.5 µM lyso-GM1 and stimulated with 2 µg/ml of CTxB or 5 µg/ml of laminin-1 for 10 minutes in the presence of 100 ng/ml of NGF. Phosphorylated Lyn (pLyn) and total Lyn were detected as described above. Bar graph shows the means +s.d. of three experiments. Relative activity, expressed as the ratio phosphorylated Lyn to total Lyn, is given as the fold-increase relative to NGF treatment (defined as 1) (*P<0.05; **P<0.01; two-sided t-test). (C) PC12 cells were transfected with siRNA targeting GM1 synthase or β1 integrin and stimulated with laminin-1 for 10 minutes in the presence of 100 ng/ml of NGF. Phosphorylated Lyn and total Lyn were detected as described above.
|
