Inhibitory effect of BtpB on phosphorylation of yeast signaling proteins.

(A) Western blotting from cells bearing the empty vector pYES2 (control), BtpA or BtpB from pYES2-GFP plasmid derivatives, developed with anti-P-MAPK antibody to detect dually-phosphorylated Slt2, Kss1 and Fus3 (upper panel) and anti-actin to detect actin as loading control. (B) Upper part: representative immunoblot from yeast cell lysates bearing pYES2-GFP-BtpB (+) or pYES2 (-) and upon different conditions: 30oC (control), high temperature (39oC), pheromone (α-factor) or Congo red, using anti-P-MAPK (upper panel), anti-Slt2 (medium panel) and anti-actin (lower panel). Lower part: densitometric measurement of WB bands corresponding to phosphorylated MAPKs Slt2, Kss1 and Fus3. The graph displays densitometric data of phosphorylated MAPKs normalized against actin and error bars show the standard deviation from three independent experiments on different transformant clones. (C) Western blotting of cells containing the pYES2 empty vector (control) or pYES2-GFP-BtpB, developed with anti-P-p38 antibody to detect MAPK Hog1 under high osmolarity. conditions (0.6M KCl). (D) Western blotting of cells expressing heterologous Akt1 (pYES3-GFP-Akt1) with either pYES2 empty vector (control) or pYES2-GFP-BtpB, using anti-P-Akt1(Thr)308 (upper panel) and anti-Akt1 antibodies. All immunoblots were performed on protein extracts from transformants of the YPH499 yeast strain after 4 h of galactose induction. (PDF)