Fig. 1.

Development of the Fer/FerT kinase inhibitor-E260 by using a yeast-based HTS system. a Native Fer (pAES-Fer) and a mutated kinase inactive Fer (pAES-FerY715F) were expressed in yeast cells using the pAES expression vector. Protein lysates from transfected cells were subjected to WB analysis using anti-HA (for HA-tagged Fer), anti-Fer, anti-phosphotyrosine (p-tyr), and anti-actin antibodies. b Growth curve of yeast cells expressing either the empty pAES vector, native Fer, inactive Fer, and non-transformed yeast cells; n = 3,±SE. c Scheme of the HTS program. d The chemical structure the 0342 compound. e The chemical structure of the E260 compound. f Growth curve of yeast transformed with the different plasmids and treated with either DMSO, 2 µM E260, or 2 µM 0342. pAES-harboring cells treated with DMSO, mutant Fer treated with DMSO, native Fer-expressing cells treated with DMSO, native Fer-expressing cells treated with 0342, native Fer-expressing cells treated with E260. Untransformed yeast cells served as a negative control; n = 3, ±SE. g Histograms depicting the 600 nm OD readings of the yeast growth-curves (presented in f) after 40 h; n = 3, ±SE. h Protein lysates prepared from yeast expressing mutant Fer and treated with DMSO for 24 h (pAES-FerY715F), active Fer and treated with 2 µM E260 (pAES-Fer E260), active Fer and treated with DMSO (pAES-Fer DMSO), or harboring the pAES vector alone and treated with DMSO, were subjected to WB analysis using anti-Fer, anti-p-tyr, and anti-PGK antibodies. Arrow on the right depicts the MW of Fer. This image presents one experiment representative of three, which gave similar results