Clone 9e10

Proteins were analysed from denatured yeast crude extracts. Briefly, 10⁸ cells/ml were harvested, resuspended in 2 ml of TCA 20% and transferred to 2 ml eppendorf tubes. The pellet was resuspended in 200 μl of TCA 20% and an equal volume of acid-washed glass beads (425–600 μm, Sigma) was added. Cells were broken by continuous vortexing for 2–4 min. 400 μl of TCA 5% was added to have a final concentration of TCA 10%. The lysates were then transferred to new 1.5 ml tubes and centrifuged for 10 min at 775×g at RT. The pellet was resuspended in 100 μl Laemmli buffer 1×. The pH was then adjusted with 50 μl of Tris Base 1 M. The protein extracts were boiled for 3 min and centrifuged for 10 min at 775×g at RT. The supernatant was collected and analysed by SDS–PAGE. Western blot acquisition and quantification was performed using BIORAD Image Lab Version 5.2.1. The following antibodies were used for WB detection: anti-FLAG (mouse monoclonal, clone M2, Sigma; cat. no: F3165, 1:5000 dilution), anti-HA (mouse monoclonal, clone 16B12, Biolegend; cat. no. 901501, 1:3000 dilution), anti-Pgk1 (mouse monoclonal, clone 22C5D8, Novex Life Technologies; cat. no. 459250, 1:5000 dilution), anti-myc (mouse monoclonal), clone 9E10, Santa Cruz Biotechnology cat. no. sc-40, 1:10,000 dilution), anti-mouse IgG HRP-linked (goat, Cell Signaling; cat. no. 7076, 1:20,000 dilution).