JCS01526 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Amino acid sequence alignment of human and mouse ARAP proteins. The human (h) ARAP1, ARAP2 and ARAP3 sequences, with the exception of the N-terminal 249 residues of ARAP1 (identified herein), have been reported previously (Krugmann et al., 2002; Miura et al., 2002). Mouse (m) ARAP3 was predicted based on sequencing of cDNA clones isolated herein. Predicted mARAP1 and mARAP2 sequences were assembled from overlapping EST and genomic sequences in the NCBI and/or Celera databases. The Clustal program was used to generate the alignment with some minor adjustments made by eye. Conserved domains (shaded boxes), amino acids conserved in all six sequences (·) and predicted tyrosine phosphorylation sites (*) are indicated.

• Supplemental Figure 2 -

  Fig. S2. Growth factors stimulate ARAP3 tyrosine phosphorylation. (A) Clonal NIH 3T3 cell lines (436-14 and 436-16) stably expressing different levels of ARAP3 were serum-starved overnight, incubated without (–) or with 20 ng/ml PDGF-BB (+) at 37°C for 20 minutes then harvested. Lysates (0.5 mg protein) were analysed by immunoprecipitation (IP) with an ARAP3 antibody, SDS-PAGE and western blotting (WB) with anti-pY (pY) and anti-ARAP3 (ARAP3) antibodies. (B) Human LNCaP cells were serum-starved, stimulated with 100 ng/ml EGF for the indicated times and then lysed. Samples of lysate (1 mg protein) were analysed as described in A. Note the two endogenous ARAP3 isoforms.