Fig. 2.

Identification of ROP18 kinase substrates using HuProt arrays. A, overall scheme to identify the ROP18 kinase substrates. Purified GST-ROP18^Δ83 and GST-ROP18^Δ83-KD proteins were assayed on HuProt arrays in kinase buffer containing [γ-³²P]ATP. After the kinase reaction, protein microarrays were washed to remove added proteins and unincorporated [γ-³²P]ATP followed by exposure to X-ray films. The films were then scanned, and bioinformatics analysis and partial validation were performed. B, representative image of the HuProt array. Individually purified human proteins tagged with GST were spotted in duplicate on a single slide, and the immobilized human proteins were visualized by probing with anti-GST antibody. C, representative photographs of X-ray films. After the kinase reactions on the HuProt arrays, the phosphorylation signals were detected by exposure of the slides to a piece of X-ray film. Developed X-ray film was then scanned, and the enlarged images of p53, MAPK11 (i.e. p38), UBE2N, and Smad1 protein dots are shown.