Ni nta sepharose

HEK293T cells were transfected with plasmids encoding T7-His-RelA, HA-ubiquitin and other tagged Set9 and WSB1/2. After 30 h of transfection, cells were treated with proteasome inhibitor MG-132 (10 μM) for 5 h and lysed in denaturing lysis buffer (20 mM Tris–HCl pH 7.9, 1% SDS) at 95°C for 5 min. Heat-denatured cell lysates were first diluted 10–20 times with dilution buffer (20 mM Tris–HCl pH 7.9, 0.5 mM EDTA, 250 mM NaCl, 2 mM NEM, 0.5% NP-40, protease inhibitor cocktail, 1 mM PMSF) to reduce the concentration of SDS and then concentrated with centrifugal filter devices (Amicon® Ultra-0.5ml, 10K). Final lysates were subjected to either Ni-NTA sepharose (GE Healthcare) for affinity purification of His-tagged RelA or HA magnetic beads for immunoprecipitation of HA-tagged ubiquitin. To measure the level of RelA ubiquitination, Ni-NTA purified RelA was immunoblotted with anti-HA antibodies; HA immunoprecipitates were immunoblotted anti-T7 antibodies.

DH–PH and DH constructs were mixed with GTPases in a 1:2 molar ratio and dialyzed into either EDTA- or MgCl₂-containing buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM DTT, and 1 mM EDTA or 10 mM MgCl₂) overnight at 4 °C. Half of the sample was then directly loaded onto an analytical Superdex75 10/300 size-exclusion column. For pull-down analysis, the samples were twofold diluted with binding buffer (25 mM Tris-HCl, pH 7.5, and 150 mM NaCl), mixed with Ni-NTA Sepharose (GE Healthcare) and incubated for 2 h at 4 °C. The slurry was transferred into a micro spin column and the unbound fraction was collected by centrifugation at 500×g for 1 min. The resin was washed with a total of 10 column-volumes buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT, 10 mM imidazole) prior to elution of the bound proteins in 3 column-volumes buffer (25 mM Tris-HCL, pH 7.5, 150 mM NaCl, 1 mM DTT, and 500 mM imidazole). The samples were analyzed on Coomassie-stained SDS-PAGE. Equal amounts of input, flow-through, wash and elution fractions were analyzed on SDS-PAGE, scanned with the Odyssey imager (Li-Cor) and quantified using the Image Studio Software (Li-Cor). The percentage of untagged protein in the respective gel lane normalized to the input fraction was calculated.

The overexpression of mouse SCGN cloned in pET21b expression vector in E. coli BL21-DE3 cells was induced with 1 mM IPTG at A₆₀₀. of 0.6 and incubated at 25 °C for 10 h postinduction as reported previously (36 (link), 57 ). Briefly, the soluble fraction of the bacterial pellet was loaded on a Ni-NTA-Sepharose (GE Healthcare) column, which was pre-equilibrated with 50 mM Tris, pH 7.5, 100 mM KCl (equilibration buffer), and washed with wash buffer (50 mM Tris, pH 7.5, 100 mM KCl, and 2% Triton X-100) followed by a wash with equilibration buffer. The protein was eluted with a gradient of 0 to 250 mM imidazole in 50 mM Tris, pH 7.5, and 100 mM KCl buffer.

In vitro kinase assay was performed as described previously (Tatarskiy et al., 2017 (link)). For in vitro kinase assay linker domain (238-361 aa), X-cluster serine mutants and NLS-3 mutant forms were cloned in pET22b vector and then recombinant 6His-linker domain proteins expressed in Escherichia coli and purified in Ni-NTA Sepharose (GE Healthcare). For the in vitro kinase experiments, 1 µg 6His-linker domain or the same amount of mutant forms were incubated with 100 µl of HEK293 extracts in the presence of gamma-31[P]-labeled ATP at 37°C. Then the samples were processed for autoradiography or western blot as specified.

Chemical reagents and buffers were purchased from Sigma-Aldrich and Biotin Coated Plates and SuperSignal WestPico Plus chemiluminescent substrate were purchased from Thermo-Scientific. Q-Sepharose and Ni-NTA Sepharose columns were purchased from GE Healthcare. Carbon/Formvar electron microscopy copper grids were purchased from Agar Scientific. AlexaFluor594 FluoroNanogold-Streptavidin was purchased from Nanoprobes.

All chemicals, reagents and enzymes were of highest quality and from Sigma‐Aldrich (St. Louis, USA), Roth (Karlsruhe, Germany) or Thermo Fisher Scientific (Waltham, USA), unless otherwise noted. Columns for affinity chromatography (Ni‐NTA‐sepharose), size exclusion chromatography (Superdex 200 10/300 GL) and buffer exchange (PD‐10 desalting column) were from GE Healthcare (Little Chalfont, UK). The E. coli strains Top10 and Rosetta™ (DE3) were from Invitrogen (Carlsbad, USA) and Merck (Darmstadt, Germany), respectively. The plasmid pET21d was from Merck (Darmstadt, Germany).