Phosphate standard

Total protein concentration from transfected HEK-293 cells lysates was determined using the Pierce™ BCA protein assay kit (Thermo Fisher scientific). Protein lysates (100 µg) were mixed with 40 µl of protein A agarose slurry (Santa Cruz Biotechnology, sc-2001) and 2 µl of anti-PP2A C subunit antibody (Sigma-Aldrich, 05-421) in a total of 500 µl with pNPP Ser/Thr assay buffer (Sigma-Aldrich, 20-179), and incubated for 2 h at 4°C with constant rotation. Next, beads were washed three times with TBS and once with pNPP Ser/Thr assay buffer. Subsequently, threonine phosphopeptide (Sigma-Aldrich, 12-219) was added to the washed beads and pNPP Ser/Thr assay buffer to a final concentration of 500 µM as substrate for the enzymatic reaction, and it was incubated for 10 min at 30°C in a shaking incubator. Twenty-five microlitres of the enzymatic reaction were mixed with 100 µl of Malachite Green Phosphate Detection Solution (solution A and additive, Sigma-Aldrich, 20–105 and 20–104, respectively) in a Corning^® 96-well half-area microplate (Merck, CLS3695-25EA), and incubated for 10 min at room temperature. Absorbance was measured at a wavelength of 650 nm in a Varioskan microtitre plate reader (ThermoFisher) and compared with the Phosphate standard (Sigma-Aldrich, 20-103).

Purified Wzc_CDΔC or Wzc_CDΔC,K540M (without the removal of the bound ADP) was exchanged into reaction buffer containing 20 mM tris (pH 7.5), 15% glycerol, 300 mM NaCl, 5 mM BME, and 50 mM MgCl₂ using spin columns to a final concentration of 3 μM. All assays were performed in triplicate. The reaction was initiated by the addition of 200 μM ATP at 37°C, following which 80 μl of aliquots was mixed with the malachite green staining solution (Sigma Aldrich) at time intervals of 10, 20, 30, 40, and 50 min. The samples were incubated for 30 min at room temperature, and the absorbance at 620 nm was measured in each case. Absorbance measurements were carried out using a 96-well plate and a SpectraMax M2 plate reader. In addition, a set of reference experiments were carried out in the absence of protein to assess background ATP hydrolysis. Inorganic phosphate concentrations were determined using the phosphate standard provided in the kit (Sigma-Aldrich) diluted into the reaction buffer.