Mouse anti pgk1

All reagents were from Sigma, except where indicated, with the following stock compositions. Yeast nitrogen base (Difco); Choline (1 M stock in DDW); Rapamycin (GoldBio; primary stock solution: 2.5 mg/ml in 100% ethanol, and secondary stock of 1 mg/ml in 90% ethanol+10% Tween‐20 (v:v), final 200 ng/ml). Copper sulfate (used at 500 μM from a 0.5 M stock in DDW). Potassium Permanganate (Dissolved in DDW), S‐1 (or Dimethylethanolamine)—Ted‐Pella−18315. BEEM embedding capsules size 00, EMS, 70010‐B. Proteinase K (stock 2 mg/ml, final 100 mg/ml (diluted in PS200)); Triton X‐100 (stock 40% (w/v), final 0.4% (w/v)); DTT (Bio Basic Inc); PMSF (200 mM in EtOH); Z1000 Zymolyase 20T (US biological); Sodium‐Sulfate (BDH); Mouse anti‐GFP (MBL); mouse anti‐Pgk1 (Abcam); Rabbit anti‐Ape1 (polyclonal antibodies prepared by using two synthetic peptides that were conjugated to keyhole limpet hemocyanin (KLH) and then injected into rabbits to produce an anti‐Ape1 antiserum that recognizes both precursor and mature forms); Anti‐cpy1 polyclonal antibody (obtained from Prof. Howard Riezman); pRS316‐GFP‐Atg8 (expressed under the promoter of Atg8, kind gift from Prof. Yoshinori Ohsumi); pDP105 (expressing TagBFP‐Ape1 under the native Ape1 promoter and Ape1 under the pCUP1 promoter, kind gift from Prof. Claudine Kraft).

Yeast total extracts were prepared as previously described (Knop et al., 1999 (link)). 1.5 × 10⁸ cells from OD₆₀₀ = 0.4–0.6 cultures were resuspended in 1150 µl lysis buffer (0.24 M NaOH, 1% β-mercaptoethanol, 1 mM EDTA, 1 mM PMSF, 5 µM Pepstatin A, 10 µM Leupeptin). After incubation on ice for 20 min, 150 µl 55% trichloroacetic acid (TCA) was added to precipitate proteins on ice for 20 min. The mixture was centrifuged at 16100 rpm at 4°C for 10 min. The pellet was resuspened in 250 µl HU buffer (8 M urea, 5% SDS, 200 mM Tris-HCl [pH 6.8], 1 mM EDTA, 5% β-mercaptoethanol, and 1% bromophenol blue) and incubated at 65°C for 10 min, followed by 16100 rpm centrifugation at RT for 5 min. The supernatant was used for subsequent analyses. Immunoblotting was performed with primary antibodies: rabbit-anti-GFP (1:2000) (Abcam), and mouse-anti-PGK1 (1:1000) (Abcam). Mouse-anti-rabbit-IgG (1:10,000) (Santa Cruz) and goat-anti-mouse-IgG (1:10,000) (Santa Cruz) were used as secondary antibodies.

Protein was extracted from 5 ml of log-phase culture using a TCA/NaOH precipitation as described in Volland et al. [57 (link)]. Equal concentrations of protein (25 μg) were run on a NuPAGE 4–12% Bis-Tris gel (Invitrogen, #NP0323BOX) and transferred to a nitrocellulose membrane (Bio-rad 0.2 µm, #LC2009). Primary antibodies used for western blotting: rat anti‐FLAG (Agilent, #200474), mouse anti‐PGK1 (Abcam, #Ab113687), mouse anti‐Rpb3 (Biolegend, #665004), rat anti-S2P (Chromotek, #3E10), rat anti-S5P (Chromotek, #3E8), Rabbit anti-Spt5-P (#1761, kind gift from Prof. Steven Hahn) [27 (link)] and Rabbit anti-Spt5 (kind gift from Prof. Grant Hartzog) [58 (link)]. Secondary antibodies used for western blotting: goat anti-mouse IRDye680RD (LI-COR, #926-68070), goat anti-rat IRDye800RD (LI-COR, #926-32219), goat anti-rabbit IRDye800RD (LI-COR, #925-32211). The Odyssey infrared imaging system (LI-COR Bioscience) was used to visualize proteins-of-interest, and signals quantified by the median method of the Odyssey software. For quantitation, data were normalized against the PGK1 signal unless otherwise stated in the figure legend.