Figure 1.

HRGP CLESH domain fusion protein specifically precipitates Vstat120 from tumor cell postculture media. A: HRGP/GST fusion proteins. Three recombinant GST fusion proteins were designed using fragments of the HRGP protein spanning amino acids 155 to 213 (within the second cystatin domain), amino acids 330 to 389 (the HRR), and amino acids 428 to 502 (CLESH domain). The linear structure of CD36 with the CLESH domain highlighted is shown below for reference. B: Coomassie stained gel (top panel) showing the purified GST-HRGP protein fragments after glutathione sepharose chromatography. The bottom panel shows Western blot analysis of each fusion protein probed with anti-GST antibody. C: Western blot analysis of a GST pull-down assay. The three GST-HRGP fusion proteins and GST alone were bound to glutathione sepharose beads, and CM from LN229V120 glioma cells were added to each sample. Each precipitate was then probed using the Vstat120/BAI1-specific antibody. Vstat120 was only detected when the CLESH domain (HRGP 428 to 502) was used in the pull-down and not the cystatin (HRGP 155 to 213) or the HRR (HRGP 330 to 389) domains. HRGP 428 to 502 only precipitated Vstat120 when CM from Vstat120-transfected LN229 cells (+ lanes) was used. No protein was detected when CM from the nontransfected parent cells (− lane) was used. This is a representative image from n = 3 experiments.