Phos tag aal

Cell lysis was carried out in MES lysis buffer (50 mM MES/NaOH pH 6.5, 150 mM NaOH, 1% Triton, 1 mM EDTA, Complete EDTA free (Roche)) by beating using a Fast Prep 24 instrument (MP Biomedicals) with the following settings: 3 × 45 s, power level 6.5. Cleared protein extracts were resolved in SDS–PAGE loading buffer (62.5 mM Tris–HCl (pH 6.8), 8 M urea, 2% (w/v) SDS, 0.05% (w/v) bromophenol blue, 10% (v/v) glycerol, 5% (v/v) β‐mercaptoethanol) and incubated at 95°C for 1 min. For Mn²⁺‐Phos‐tag SDS–PAGE, 25 µM of Phos‐tag‐AAL (Wako) and 50 µM of MnCl₂ were added to the separating gel before polymerization. The pH of the separation gel was adjusted to 8 for Mn²⁺‐Phos‐tag SDS–PAGE and 8.8 for standard SDS–PAGE. Gelshifts were visualized by Western blot using an antibody recognizing HA (12CA5). Hyperosmotic stress was controlled by monitoring phosphorylation of MAPK Hog1 using an antibody against phospho‐p38 (Cell Signaling, Phospho‐p38 MAPK (Thr180/Tyr182) Antibody #9211, 1:5,000), protein levels of Cdc55 by using an antibody against Cdc55 (Max Perutz Labs Vienna, Monoclonal Antibody Facility, 1:200 (Zuzuarregui et al, 2012 (link))). Loading was controlled using an antibody recognizing Cdc28 (HPA030762, Sigma‐Aldrich, 1:10,000).

Cell pellets were resuspended in SDS loading buffer (1×) and boiled at 100°C for 10 minutes. SDS-PAGE gels supplemented with 50 μM MnCl₂ and 50 μM Phostag AAL (Wako) were used to separate proteins. When the dye front escaped, the gels were washed for 10 minutes in transfer buffer supplemented with 1 mM EDTA. After 3 wash steps, the gels were transferred onto nitrocellulose membranes (66485, Pall), and standard Western blotting was performed to detect phosphorylated proteins.

Phos-Tag Acrylamide gel was realized adding to resolving gel 50 μM Phos-Tag™ AAL (FUJIFILM Wako Chemicals U.S.A. Corporation) and 10 mM MnCl₂. Then Phos-Tag gel was subjected to conventional SDS-PAGE. Prior to electro blotting the gel was washed twice in 1 mM EDTA dissolved in water and then in 1 mM EDTA dissolved in Blotting Buffer. Then WB was performed as described in the WB section.