Protran 0.45 μm

For western blot analyses cells were collected 0, 6, 8, 12, 16, and 24 h after treatment, respectively. The cells were taken up in lysis buffer and stored at −80°C as described before (Grabiec et al., 2019 (link)). Protein concentrations were determined using the method of Bradford (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Barrington IL, United States) (Bradford, 1976 (link)) and 10 μg protein was loaded onto a 12.5% (w/v) sodium dodecyl sulfate–polyacrylamide gel. After gel electrophoresis, the proteins were blotted onto nitrocellulose membranes (Protran 0.45 μm, Amersham, Freiburg, Germany) and non-specific protein binding sites were blocked for 30 min with roti block solution (Carl Roth, Karlsruhe, Germany). The nitrocellulose membranes were incubated with the antibody against IMPDH2 (Table 1) [diluted 1:2000 in roti block solution containing 0.2% (v/v) Tween 20] until the next day at 4°C. After incubation with horseradish peroxidase conjugated antibodies (Table 1) the chemiluminescent (Luminata Forte, Merck) signal was detected with Fusion X (VWR, Radnor, PA, United States). For semi quantitative analysis, the relative signal intensities of the immunoreactive bands were determined and the IMPDH2 intensity was normalized to the ß-actin (Table 1) intensity. Fusion FX7 with FusionCapt Advance Solo software (VWR) was used for analysis.

Purified Noc (WT and variants) (final concentration: 30 μM) were incubated with 5 nM radiolabeled P³²-α-CTP (Perkin Elmer), 30 μM of unlabeled CTP (Thermo Fisher), 1 μM of 22 bp NBS or parS DNA in the reaction buffer [100 mM Tris pH 8.0, 100 mM NaCl, and 5 mM CaCl₂] for 5 min at room temperature. For the NTP competition assay, the mixture was further supplemented with 500 μM of either unlabeled CTP, CDP, ATP, GTP, or UTP. Four μL of samples were spotted slowly onto a dry nitrocellulose membrane (Amersham Protran 0.45 μm) and air-dried. Subsequently, membranes were exposed to a phosphor screen (GE Healthcare) for two minutes. Each DRaCALA assay was triplicated, and a representative autoradiograph was shown.