Ficity of the Slit2 antibody for each human and rat Slit2. c: Coomassie Blue stain of purified rhSlit2 and RoboN using immobilized immunoaffinity chromatography. a: Alignment with the human peptide sequence (major) with rat Slit2 (bottom) showing 95 homology. This human peptide sequence was used to generate the antiserum made use of in rats. b: Anti-human Slit2 antiserum was employed in Western blotting experiments (at 1 in 5000) and was able to detect each human (lane 3) and rat (lane four) Slit2 from transfected 293T cells. A single band of approx 230 kd was observed. Wild-type 293T cells (lane 1) and 293T cells transfected with vector alone (lane two) didn’t show proof of Slit2 expression. c: Lane 1, protein size markers (M); lane 2, 1 g of rhSlit2; lane three, 1 g of RoboN. A prominent band representing rhSlit2 (slit) was observed at 220 kd (second lane, open arrow). A smaller sized, less prominent protein species was also observed at one hundred kd. As observed in Figure 1B, this was not detected by the slit antibody; RoboN was seen as a single smear of molecular weight among 85 and 95 kd, possibly as a consequence of the heavy glycosylation (third lane, black arrow).400 l, have been electroporated (at space temperature, 300V for 25ms) with 20 g of plasmid (pcDNA3.1) containing either the human or rat Slit2 sequence. Controls were also performed using the vector alone. Electroporated cells had been recovered in 20 fetal CLEC2B Proteins Synonyms bovine serum (FBS) Dulbeccos modified Eagle’s media (DMEM) medium at 37 for 24 hours. By Western blotting, bands in the suitable size ( 220 to 240 kd) were seen in the 293T cells transfected with Slit2 but not inside the control cells (vector alone, Figure 1B).Production of rhSlit2 and RoboNBoth rhSlit2 and RoboN were created from stably transfected 293T cells. The strategies necessary have been extensively described previously.five,6 The full-length human Slit2 cDNA sequence plus the extracellular domain of Robo1 had been tagged at the carboxy terminus with c-myc and HA, respectively. Cells were cultured in DMEM supplemented with five fetal bovine serum and media was collected 3 days right after cells became confluent. In short,344 Kanellis et al AJP July 2004, Vol. 165, No.slit- and