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. Author manuscript; available in PMC: 2016 Sep 10.
Published in final edited form as: Cell. 2015 Sep 10;162(6):1286–1298. doi: 10.1016/j.cell.2015.08.041

Figure 3.

Figure 3

Heat-triggered protein aggregation does not require ongoing translation. A, Cycloheximide (CHX; 100 µg/mL) blocks formation of heat-triggered cytosolic foci by fluorescently tagged Pab1 and attenuates formation of foci by Yef3. Scale bars are 5µm, arrows indicate foci. B, Ola1 forms fluorescent foci in response to heat shock in the presence of CHX. C, Pab1, Ssz1, and Yef3 (arrows on gel) are found in the 100,000 g supernatant (S) in lysate from unshocked cells, but enter the 100,000 g pellet (P) after heat shock independent of CHX treatment. Coomassie-stained protein gel and western blots against native proteins are shown. T, total protein. D, CHX inhibits Pab1 and Ssz1 entry into large aggregates, but not into small aggregates. Cell lysate was progressively fractionated at 8,000 g (pellet, P8), 20,000 g (P20), then 100,000 g (P100), and pellets and residual supernatant (S) were western blotted against Pab1 and Ssz1; intensity as proportion of total was quantified in two biological replicates (Fig. S6), and a representative blot is shown.