Isolating Myo2p in the absence of functional Rng3p.

<p>Full-length over-expressed Myo2p motors were purified from wild-type (<i>rng3⁺ nmt41^prom-myo2</i>) and mutant (<i>rng3-65 nmt41^prom-myo2</i>) cells following growth at the permissive (25°C) or restrictive (37°C) temperature. A) Western blot depicting Myo2p levels (upper panel) from whole cell lysates following induction of Myo2p over-expression (upon removal of thiamine, time zero) in <i>rng3-65</i> cells. Cells were shifted to the restrictive temperature at 16 hours post-induction, before any appreciable Myo2p expression is detected by antibody (upper blot). The lower blot shows loading controls (Hsp90 levels) for each sample. Protein samples were normalized based on total protein concentration of the lysates. The plot shown below tracks relative levels of Myo2p detected versus time (post-induction). Relative density values for the Myo2p bands were corrected using densities from accompanying Hsp90 bands; final values were normalized whereby 1 is equal to the maximum value. B) Western blots to determine the relative levels of Myo2p produced in the different isolations. Blots were employed (instead of Coomassie staining) to clearly distinguish the Myo2p heavy chain bands in these crude samples. Upper blot: α-Myo2p depicting the relative levels of Myo2p 1-step purified from cells shifted to 37°C (or retained at the permissive growth temperature in accompanying controls) at 16 hours post-induction. The levels shown are from samples harvested at 28 hours post-induction. Samples were diluted 100-fold prior to loading on gels. Lower blot: loading control showing a non-specific band detected by α-Hsp90 antibodies in undiluted samples. Myo2p concentrations were equalized based on the relative levels observed in these blots (prior to testing of the different samples in motility assays; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079593#pone-0079593-t001" target="_blank">Table 1</a>).