Goat anti mouse hrp conjugated secondary antibody

Strains were grown at 30°C in YPD to reach OD600 = 0.4–0.6 and harvested by centrifugation. Cell extracts were prepared by resuspending the pellets in cold buffer A (50 mM HEPES, 0.4 M (NH₄)₂SO₄, 1mM EDTA, 5% glycerol) with protease inhibitors and bead beating at 4°C for 2 min four times, with 2 min breaks on ice between bead beating rounds. Lysate was removed from beads and Triton X- was added to a final concentration of 1% before centrifuging for 30 min at 20,000 g and 4°C. Total protein (the supernatant) was denatured in 1X SDS running buffer (1X NuPage LDS sample buffer, 10% β-mercaptoethanol) with protease inhibitors by heating at 95°C for 10 min and then chilled on ice for 2 min. Subsequently, 30 μL of the denatured protein extract was separated by SDS-PAGE using an Invitrogen Bolt 4–12% Bis-Tris acrylamide gel and transferred onto a 0.2 μm nitrocellulose membrane. Immunoblots were developed using the Pierce Supersignal Femto Kit and imaged with a Bio-Rad ChemiDoc XRS+ system. To detect Msn2 constructs, we incubated at 4°C overnight with 1:1000 mouse anti-RFP antibody (Chromotek 6G6) and incubated at room temperature for 1 h with 1:20,000 goat anti-mouse HRP-conjugated secondary antibody (ab97023). Bands were quantified in ImageJ following recommended practices.^{51 (link),57 (link)}

Surface biotinylation and immunoblot analysis were performed following the procedures previously described.^{28 ,29 (link)} Briefly, cell surface proteins were labeled with sulfo-N-hydroxysuccinimide-SS-biotin and isolated by streptavidin-agarose beads. Isolated membrane proteins were then subjected to SDS-PAGE and Western blotting. Transporters were detected with a mouse anti-His tag antibody (Proteintech) (1:20000), followed by an HRP-conjugated goat anti-mouse secondary antibody (Proteintech) (1:5000). The plasma membrane marker Na⁺/K⁺-ATPase was detected with a rabbit anti-Na⁺/K⁺-ATPase α subunit antibody (Abcam) (1:10000), followed by an HRP-conjugated mouse anti-rabbit secondary antibody (Proteintech) (1:10000).