Fig. 3
From: Light regulates the degradation of the regulatory protein VE-1 in the fungus Neurospora crassa
The degradation of VE-1 by the proteasome is regulated by light. a Regulation by light of VE-1 degradation. Cultures of vegetative mycelia of the ve-1FLAG and the Δwc-1 ve-1FLAG strains were grown at 30 °C in liquid media for 24 h in the dark, light or exposed to 30 min of light, then cycloheximide was added to the cultures, and samples were collected at different times. Proteins were separated by SDS-PAGE, and hybridized with an antibody specific for FLAG. Seventy micrograms of proteins was loaded per lane. As loading control, we used a Ponceau staining of each protein sample. Each hybridization was quantified using as reference the amount of VE-1 detected at time point 0 (before addition of cycloheximide). The plot shows the average and standard error of four independent experiments. b The proteasome inhibitor thiolutin prevents the degradation of VE-1. Cultures of vegetative mycelia of the ve-1FLAG strain were grown at 30 °C in liquid media and exposed to 30 min of light, then cycloheximide and/or thiolutin were added to the cultures, and samples were removed at different times. Cultures without any additional chemical added were used as controls. Proteins were separated by SDS-PAGE, and hybridized with an antibody specific for FLAG. Seventy micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. The hybridization was quantified using as reference the amount of VE-1 detected at time point 0 (before addition of cycloheximide, thiolutin or both). The plot shows the average and standard error of three independent experiments. c FWD-1 and the CSN participate in the degradation of VE-1. Cultures of vegetative mycelia of the Δcsn-1 ve-1FLAG, Δcsn-5 ve-1FLAG, and Δfwd-1 ve-1FLAG strains were grown at 30 °C in liquid media for 24 h in the dark, light, or exposed to 30 min of light, then cycloheximide was added to the cultures, and samples were removed at different times. Proteins were separated by SDS-PAGE and hybridized with an antibody specific for FLAG. Seventy micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. Each hybridization was quantified using as reference the amount of VE-1 detected at time point 0 (before addition of cycloheximide). The plot shows the average and standard error of 2–3 independent experiments