Slides and culture plates were coated with poly lysine or lam inin, where appropriate. The neurons were cultured in modified serum free NB alone with no added growth factors. Immunocytochemistry Neurons were fixed selleck catalog in 4% paraformaldehyde in PBS for 20 minutes, permeabilized with 0. 1% Triton X 100 and blocked with 5% normal goat serum in PBS. Anti bodies used were as follows Hsp27 and phospho Hsp27S15, total tubulin, actin. It should be noted that the Hsp27 antibody recognizes both the non phosphorylated and phosphorylated Hsp27, while the pHsp27 antibody only recognizes the phosphorylated form. We have also tested two other pHsp27 antibodies, but have found the Affin ity Bioreagents Antibody to be better for immunostaining. Cells were incubated with the primary antibodies at 4 C for 16 20 hrs, followed by Cy2 or Cy5 tagged secondary antibodies.

In some experiments, cells were also labelled with rhodamine phalloidin after antibody incubation. The slides were coverslipped with glycerol and imaged with confocal laser scanning microscopy using z stage scanning and image stacking. Stacked digital images were imported into Adobe Photoshop for compilation into the final composite figures. Laminin stimulation and neurite growth initiation Neurite initiation was assessed in two ways. The first series of experiments employed neurons plated on laminin coated slides, with the cells being fixed and analyzed for outgrowth parameters at 24 hrs after plating. In a second series of experiments, the neurons were first plated on polylysine coated slides and allowed to stabilize overnight prior to being stimulated with soluble laminin.

Following the addition of the laminin solution, cells were then fixed at 5, 15, 30 min, 1 hr, 6 hr, and 24 hr. After fixation, cells were immu nostained and analyzed as described above. Inhibitor experiments SB 203580 and SB 202190 were used to inhibit p38 MAPK activity, in order to assess the contribution of phos phorylated Hsp27. Inhibitors were added 1 hr prior to laminin stimulation. For the 24 hr Cilengitide cultures, the inhibitors were added 2 3 hrs after plating the cells on laminin coated slides and retained in the medium for the extent of the experiment. Cytochalasin D was also used in longer term experiments, and was added 3 hrs after plating and maintained in the medium for the extent of the experiment Immunoblotting For Western analyses, neurons were plated in 12 well plates that had been coated with polylysine alone or with laminin, depending on which experimental paradigm was used. Neurons were subsequently processed according to our established procedures. Cellular fractionation was carried out using a subcellular protein extraction kit to isolate cytoplasmic, mem brane, nuclear and cytoskeletal fractions.