Fig. 8.

MoSsb1, MoSsz1, and MoZuo1 are required for the maintenance of protein amounts of MoMkk1. A, Guy11, ΔMossb1, ΔMossz1, and ΔMozuo1 strains containing MoMkk1-GFP (green fluorescent protein) were grown in liquid complete medium for 48 h. Total proteins extracted from the strains were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots were incubated with anti-GFP. Protein amounts of MoMkk1-GFP in DMossb1, ΔMossz1, and ΔMozuo1 were significantly reduced. B and C, The Western blotting bands detected with anti-GFP were quantified with the ODYSSEY infrared imaging system (application software version 2.1). The intensity of the MoMkk1-GFP band for each strain is relative to that of total protein. Bars denote standard errors from three independent experiments. Values on the bars followed by the same letter were not significantly different at P = 0.05. D, Transcription analysis of MoMKK1-GFP in Guy11 and three mutants containing MoMkk1-GFP using quantitative reverse transcription-polymerase chain reaction. E, MoMkk1-GFP from the wild-type strain, ΔMossb1, ΔMossz1, and ΔMozuo1 mutants cell extracts treated with alkaline phosphatase or phosphatase inhibitors were subjected to Phos-tag SDS-PAGE and normal SDS-PAGE followed by immunoblotting with anti-GFP.