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. 2018 Dec 27;13(1):21–32. doi: 10.1080/19336896.2018.1558763

Figure 1.

Figure 1.

Toh1-YFP protein forms detergent-resistant aggregates in yeast cells that are revealed predominantly in the cell debris. (a) Localization of the Toh1-YFP aggregates in yeast cells. Transformants of BY4742 strain expressing TOH1-YFP and GFP genes were grown on -Leu selective media for 48 h prior to fluorescence microscopy. In the case of the constructs under PCUP1 promoter media was supplemented with 100 μM CuSO4. (b) Fractionation of the crude cell lysates extracted from BY4742 cells expressing the Toh1-YFP protein showing predominating localization of Toh1-YFP in the debris fraction. Cells expressing Toh1-YFP protein were harvested after 48h, lysed and the crude cell lysate was separated into debris and lysate fractions followed by the denaturating treatment of the samples (2,5% SDS, 95°C). Samples were resolved in 10% SDS-PAGE and immunoblotted using anti-GFP antibody (ab32146 (Abcam, Great Britain)). (c) SDD-AGE of the crude protein lysates extracted from BY4742 cells expressing Toh1-YFP protein showing the capacity of Toh1-YFP to form SDS-resistant aggregates. The debris fractions of the crude protein lysates were incubated under semi-denaturing (SD, 1% SDS at room temperature) and denaturing (D, 2,5% SDS, 95°C) conditions. Samples were resolved on 1,5% SDS-AGE and immunoblotted using anti-GFP antibody (ab32146 (Abcam, Great Britain)). Cells expressing GFP were used as a negative control for aggregation.