Figure 5.

Sik SH2 domain binds directly to GAP-associated p65 in an overlay assay. Precipitations were done with keratinocyte lysates at 10 min after Ca²⁺ addition by using anti-GAP antibody or GST or the GST–Sik SH2 domain. Precipitates were washed and split into four samples that were subjected to SDS/PAGE and transferred to Immobilone-P membranes. Filters were probed with anti-phosphotyrosine antibody (A), anti-GAP antibody (B), biotin-labeled GST (C), or biotin labeled GST–Sik SH2 domain fusion protein (D). Primary antibodies were detected as described. Bound GST and GST fusion proteins were detected by preformed avidin and biotinylated horseradish peroxidase complex and ECL.