Pgk1 22c5d8

The yChEFs procedure was followed to prepare chromatin-enriched fractions [88 (link)] using 50 mL of exponentially grown YPD cultures (OD600~0.6–0.8). The final chromatin-bound proteins were resuspended in 1X SDS-PAGE sample buffer, boiled and analysed by western blot with different antibodies.
Protein electrophoresis and western blots were carried out as described in [50 (link)]. The employed antibodies included the anti-Rpb1 (against the CTD; manufactured in our laboratory) [88 (link)], anti-RNA pol II CTD phosphor Tyr1 (Active Motif), anti-RNA pol II phospho-CTDSer7 (4E12) (Chromotek, Planegg, Germany), anti-phosphoglycerate kinase, Pgk1 (22C5D8; Invitrogen, Massachusetts, USA ), anti-H3 (ab1791; Abcam, Cambridge, UK) and PAP (Sigma, St. Louis, MO, USA) antibodies.
IMAGE STUDIO LITE software (5.2. version) was utilised to quantify the intensities of the immunoreactive bands.

Protein electrophoresis and Western blot were carried out as described in [62 (link)].
For the Western blot analyzes, anti-CTD [63 (link)], anti-Rpb3 (anti-POLR2C;1Y26, Abcam), anti-Rpb4 (Pol II RPB4 (2Y14); Biolegend, San Diego, CA, USA), anti-Rpb5 (a polyclonal antibody generated against S. cerevisiae Rpb5 in our lab), anti-Rpb6 (a gift from M. Werner), anti-Rpb7 (Rpb7 (yN-19); Santa Cruz Biotechnology, Dallas, TX, USA), antiphosphoglycerate kinase, Pgk1 (22C5D8; Invitrogen), anti-H3 (ab1791; Abcam), antihemaglutinin (anti-HA; 12CA5; Roche) and PAP (Sigma) antibodies were used.
Intensities of immunoreactive bands on Western blots were quantified by densitometry using the software IMAGE STUDIO LITE from images acquired with an office scanner.

Autophagy progression was monitored by the immunological detection of the Gfp accumulation processed from Gfp–Atg8, which is delivered to the vacuole to be degraded upon autophagy induction [35 (link)]. Gfp moiety is very resistant to proteolysis compared to Atg8. For these assays, the WT and bud27Δ cells were transformed with the pRS315-GFP-ATG8 (CEN; LEU2) plasmid to allow the expression of Gfp-Atg8. Cells were grown at 30 °C in SD minimal medium to the mid-log phase (OD₆₀₀~0.6), which corresponded to experiment time 0. Then, cells were washed three times, diluted, and shifted to SD (-nitrogen) during a time course (up to 2 h). Proteins were precipitated with TCA from 1 mL of culture cells and analyzed by western blot using an anti-Gfp antibody (GFP (D5.1), 2956; Cell Signaling). Anti-phosphoglycerate kinase, Pgk1 (22C5D8; Invitrogen, Waltham, MA, USA) was used to detect Pgk1 as the internal control.
The intensities of the immunoreactive bands on western blots were quantified by densitometry using the IMAGE STUDIO LITE software from the images acquired with an office scanner.