MID1 contains phosphorylated serine and threonine residues. (A) Extracts from untransfected Cos1 cells (lanes 1 & 3) or Cos1 cells transfected with GFP-MID1 (lanes 2 & 4) were immunoprecipitated with anti-GFP antibody/protein-A sepharose beads and analysed by western blot analysis using either an anti-phosphoserine antibody (lanes 1 & 2) or anti-phosphothreonine antibody (lanes 3 & 4). Protein bands in lanes 2 and 4 indicated that GFP-MID1 contains both phosphoserine and phosphothreonine residues. (B) Domain-specific deletions of MID1 were used in an attempt to crudely map locations of the phosphorylated serine and threonine residues. Shown are extracts from Cos1 cells transfected with full-length GFP-MID1 (lane 1), GFP-MID1ΔRF (lane 2), GFP-MID1ΔBB (lane 3), GFP-MID1ΔCC (lane 4), GFP-MID1ΔFNIII (lane 5), and GFP-MID1ΔCTD (lane 6). The samples were immunoprecipitated with anti-GFP antibody/-protein-A sepharose beads, separated on 8% SDS polyacrylamide gels, transferred to nitrocellulose membranes and blotted with anti-phosphoserine antibody (top panel) or anti-phosphothreonine antibody (bottom panel). (C) Computer assisted detection of potential serine/threonine phosphorylation sites in MID1. Potential serine/threonine kinase consensus phosphorylation sites in MID1 were identified using NetPhos 2.0 software . Examination of a multiple alignment of available MID1 and MID2 sequences was carried out to identify fully conserved putative phosphorylation sites. A diagrammatic representation of this analysis shows 16 conserved sites depicted as dots (red for serine and blue for threonine) along the length of a representative MID1 protein (numbered residue positions are also depicted at the top of diagram). The actual kinases that recognise the residues are listed on the left of the figure; CaMII (R-X-X-S/T-X), CKI (Sp/Tp-X2–3-S/T-X), CKII (X-S/T-X-X-D/E), GSK3 (X-S/T-X-X-X-Sp), MAPK (P-X-S/T-P), PKA (R-X1–2-S/T-X), PKC (X-S/T-X-R/K), PKG ((R/K)2–3-X-S/T-X).