Cell lysates from logarithmically growing ppn1Δppn1Δppx1Δ (Δ3), a triple polyphosphatase mutant and the isogenic vtc4Δ mutant ppn1Δppn1Δppx1Δvtc4Δ (Δ4), a mutant where polyP is absent, were prepared using lysis buffer containing either 150 or 500 mM NaCl and resolved on NuPAGE. Western blot using anti-Nsr1 antibody reveals a considerable mobility shift of Nsr1 in the triple polyphosphatase mutant strain (Δ3), while this mobility shift is absent in the isogenic vtc4Δ mutant (Δ4). Under high salt (500 mM) condition, the dehydrated shrunken vacuole10 is not lysed, leading to a smaller Nsr1 mobility shift resulting from the modification by the less-abundant and shorter nuclear polyP.9 An identical mobility shift is observed by adding exogenous polyP100 (+) to the Δ4 lysate, prepared with either 150 or 500 mM NaCl lysis buffer. Ponceau stain is used to verify equal loading. The data presented are representative of 2 independent experiments.
