Figure 3.

Dephosphorylation of NF by PP2A is inhibited by addition of Pin1 in vitro. A, NF triplet (NF-H/M/L) proteins were prepared from rat spinal cord and sciatic nerve as described in Material and Methods and was subjected to dephosphorylation with 1 U of PP2A (lane 2). The effect of Pin1 on NF-H dephosphorylation was tested by preincubating 2.5 μg of NF-triplet protein with GST–Pin1 (0.25 and 0.5 μg; lanes 3–4) at 30°C for 10 min, followed by addition of 1 U of PP2A. Reaction mixtures were incubated at 30°C for 30 min. Reactions were terminated by addition of SDS loading buffer, and proteins were analyzed by 4–20% SDS-PAGE. Blots were probed with monoclonal antibodies for phosphorylated NF-H (RT97). Data show the effect of Pin1 on NF-H dephosphorylation. PP2A dephosphorylates NF (lane 2). Addition of Pin1 prevents NF dephosphorylation in a dose-dependent manner [lane 3 (0.25 μg of Pin1); lane 4 (0.5 μg of Pin1)]. B, Densitometry analyses of phospho-NF obtained from A.