ZC3H30 is in a granule fraction after heat shock.

A) In procyclic trypanosomes grown at 27°C, ZC3H30 is in the cytosolic fraction. Trypanosomes were disrupted using silicon carbide, and fractionated by centrifugation. 5 x 106 cell-equivalents were loaded on each lane of an SDS-PAGE gel and the proteins were detected by Western blotting. Tot: total lysate; C: cytosol: Sup: detergent-soluble organellar proteins; Pel: IGEPAL-insoluble pellet. TR is trypanothione reductase, a cytosolic protein, and XRND is in the nucleus. (B) ZC3H30 moves to the granule fraction after heat shock. Trypanosomes expressing V5-ZC3H30, with or without a 1h heat shock at 41°C, were fractionated into various soluble and granular fractions, using the cytoskeleton as a molecular sieve to trap stress granules. T: Unfractionated total cell lysate; SN1-3: soluble supernatants; SN4: molecules trapped inside cytoskeletons but not sedimented at 20000g (10min); G: sedimentable granules. Proteins were detected from equal proportions of each fraction. DHH1 and SCD6 are stress granule markers and trypanothione reductase (TR) is in the soluble fraction. (C) Loss of GFP-Tb927.8.3820 after 24h RNAi induction in procyclic forms (control for panel (D)). The Ponceau protein stain serves as a loading control. (D) ZC3H30 is in the granule fraction in heat-shocked Tb927.8.3820-depleted cells. As in (B), but using cells with RNAi against Tb927.8.3820 (see (C)).