Molecular weight standards

The wild-type VP and five selected variants were isolated from 500 mL culture medium by centrifugation at 3000 × g for 5 min at 4 °C, followed by resuspending the cell pellet in 50 mL 100 mM sodium acetate (pH 5.5) containing 1 mM 2-mercaptoethanol. The suspension was incubated at 4 °C for 4 h before repeating the centrifugation step [55] (link). The supernatant containing Aga2-VP fusion proteins was concentrated using Vivaspin ultrafiltration columns with a J o u r n a l P r e -p r o o f molecular weight cutoff of 10 kDa (Sartorius-Stedim, Göttingen, Germany) and the concentrated supernatants were dialyzed against 20 mM sodium phosphate buffer (pH 7.4). The proteins were purified using Vivapure Q Mini H mini spin columns (Sartorius-Stedim). Fractions containing active Aga2-VP variants were collected and analyzed by denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 12% gels containing 2-mercaptoethanol.
Protein bands were revealed by staining with Coomassie Brilliant Blue R-250 and compared to molecular weight standards (Thermo Fisher Scientific, Waltham, MA, USA). Native electrophoresis was performed under the same running conditions in gels without SDS and 2-mercaptoethanol. For zymography, the gel was supplemented with 100 mM sodium tartrate (pH 3.5) containing 0.5 mM H2O2 and 9 mM guaiacol [55] (link).

D-raffinose, 2, 3, 5-triphenyl tetrazolium chloride, D-galactose, ampicillin, SDS, 2',7'dichlorofluorescin diacetate (DCFDA) and lithium acetate were purchased from Sigma (USA). D-glucose was procured from Amresco (USA). Yeast nitrogen base, yeast extract, peptone, DMSO, chloroform, agar and glass slides were procured from HiMedia (India).
Salmon sperm DNA was purchased from Invitrogen (USA). CellRox Deep Red reagent (catalogue no: C10422) was purchased from Thermofisher scientific. For western blot, CellLytic TM Y (Sigma USA), Bradford reagent (BioRad), Western blot blocking buffer (Himedia India), anti-TDP-43 primary antibody (Sigma, catalogue no: WH0023435M1), anti-GAPDH primary antibody (Abcam, catalogue no: ab125247), anti-mouse secondary antibody tagged with alkaline phosphatase (Sigma, catalogue no: A3562), CDP star (sigma), and molecular weight standards (Thermofisher scientific), were used. Propidium iodide (Sigma, catalogue no: P4170) was used for dead cell staining. For staining the yeast nuclei, DAPI (Sigma, catalogue no: D9542) or Hoechst 33342 (Thermofisher scientific, catalogue no: 62249) were used.