Flag specific

Complemented strain C. glutamicum 14067-△amtR:amtR-3Flag was grown in BHI medium, and AmtR-3Flag production was induced using 0.5 mM IPTG (isopropyl -β-d-thiogalactopyranoside) for 8 h. AmtR-3Flag production was not induced with 0.5 mM IPTG as the negative control. The cells were harvested by centrifugation and washed with PBS buffer (150 mM NaCl, 3 mM KCl, 10 mM Na₂PO₄, 3 mM KH₂PO₄, pH 7.5), suspended in PBS buffer to normalize the culture densitiy based on the OD₆₀₀ value, and disintegrated with silica beads (0.1 mm) for 12 cycle of 30 s at a speed rating of 6.0 with 3 min resting intervals by Bead Ruptor 12 (OMNI International, United States). Soluble extracts were fractionated on a 12% denaturing polyacrylamide gel before being transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, United States). The membrane was blocked with 3% bovine serum albumin and incubated overnight at 4°C with a 1:2,000 dilution of Flag-specific (Sigma-Aldrich, St. Louis, MO, United States) mouse antiserum, and incubated with a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TX, United States). Finally, the immunoreactive protein bands were visualized with an ECL reagent (Thermo Fisher Scientific Inc., Waltham, MA).

Immunoprecipitation of Ubp2^C745S-Flag was performed as follows: 160 OD₆₀₀ of yeast cells grown in SCD media to the exponential growth phase were disrupted with glass beads (0.4–0.6 µm) in RIPA buffer without detergents (HEPES-KOH 40 mM pH 7.6, NaCl 150 mM, EDTA 5 mM). After centrifugating at 16000 g for 10 min, the supernatant was diluted in an equal volume of RIPA buffer containing 2X detergents, so that the final composition was HEPES-KOH 40 mM pH 7.6, NaCl 150 mM, EDTA 5 mM, Triton X100 1%, SDS 0.1%, sodium deoxycholate 0.5%. After sonication for 15 min at 4°C in a water bath, denatured cytosolic fractions were employed to precipitate Ubp2^C745S-Flag. Flag-coupled beads (Sigma-Aldrich) were used for overnight immunoprecipitation and protein elution was performed with Laemmli buffer for 20 min shaking at 40°C. The eluate was split in two and resolved in 8% Tris-glycine gels. After transfer, the nitrocellulose membrane was divided in two: one half of the eluate was immunoblotted with a Flag-specific (Sigma) and the other half with a ubiquitin-specific antibody (P4D1; Cell Signaling).