Fig. 4. (A) The phosphorylated regulatory subunit of PI3K, pTyr-p85, is associated primarily with the growth cone cytoskeleton. Western blots of GCP subfractions (S, cytosol; M, membranes; and C, cytoskeletal fraction). GCPs were preincubated in control medium, with IGF-1 (10 nM, 5 minutes) or with BDNF (0.2 nM, 5 minutes). Blots were probed with anti p85 (middle) or anti-pTyr-p85 (top and bottom). Equal amounts of protein were loaded in each lane. Note the significant activation of PI3K with IGF-1 (top), but not with BDNF (bottom). pTyr-p85 was highly enriched in the cytoskeletal fraction and, to a lesser extent, in the membrane fraction. (B) Western blot of GCP polypeptides probed with anti-p110 antibody. Note mono-specificity of the antibody.