Mouse anti pgk1 antibody

For yeast, 1 OD₆₀₀ of mid-log phase cells was collected by centrifugation and precipitated using 10% trichloroacetic acid for 20 min at 4 °C. After centrifugation at 13,000g for 5 min, pellets were washed with ice-cold acetone. Pellets were air-dried and resuspended in 30 µl of 1× SDS sample buffer (60 mM Tris pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.005% bromophenol blue), and boiled for 3 min. For mammalian cells, 10⁶ cells were scraped off in 100 µl of SDS sample buffer and heated at 96 °C for 10 min. Samples were resolved on a 12% SDS–PAGE gel, and after transfer on a PVDF membrane, proteins were detected using specific antibodies. The following antibodies were used: mouse anti-Pgk1 antibody (Invitrogen, 459250, 1:3,000 dilution), rat anti-RFP antibody (Chromotek, 5F8, 1:1,000 dilution), mouse anti-FLAG antibody (Sigma, F1804, 1:1,000), rabbit anti-mCherry antibody (Abcam, ab167453, 1:1,000) and horseradish-peroxidase-coupled secondary antibody (Bio-Rad, 170–6516; 1:10,000 dilution). Western blots were imaged using the Fusion FX system (Vilber) equipped with the FusionCapt Advance FX7 software (version 17.03).

For the preparation of total cell lysates in Fig. 6A, about 10 OD₆₀₀ units of cells at midlog phase (OD₆₀₀ = 0.8 to 1.0) were harvested in 1.5 mL tubes, flash frozen in liquid nitrogen, and stored at −80°C. The cells were treated using a cell wall loosening procedure (2 M LiAc on ice for 3 min followed by 0.4 M NaOH on ice for 3 min),²¹ (link) and disrupted in 50 mM Tris (pH 8.0), 1% SDS, 8 M urea (VWR, 28877), 2 mM DTT, 5 mM EDTA with glass beads at 3,000 rpm for 30 sec with PowerLyzer homogenizer (MO BIO Laboratories, 13155, Carlsbad, California, USA) at 4°C. One volume of 4X sample buffer was added to 3 volumes of the lysates and loaded onto the gel. Proteins were detected using a mouse anti-Pgk1 antibody (Invitrogen, 459250) with anti-mouse-IgG HRP (Sigma, A9917), or an anti-Flag M2-HRP (Sigma, A8592). For detecting the His-tagged human NRBF2 MIT domain, Penta-His HRP conjugate Kit (Qiagen, 34660) was used with BenchMark (Invitrogen, 10747012) as a marker.