Imagequant tl

Purified His-SUMO-EssS(191–488), CenR, and EssR were diluted to 5 μM with 1x kinase buffer (250 mM Tris pH8, 50 mM KCl, 10 mM MgCl2, 10 mM CaCl2, 1 mM DTT). 5 μl of ATP mix (4.5 μl 250 μM ATP, pH 7, 0.5 μl [32P]-ATP (10 μCi/μl) was added to 45 μl of 5 μM His-SUMO-EssS and the protein was allowed to autophosphorylate for 1h at room temperature. 5 μl of His-SUMO-EssS~P was mixed with 5 μl 1x kinase buffer and SDS loading buffer as the autophosphorylation control. To assess phosphoryl transfer, His-SUMO-EssS~P was mixed with equimolar of CenR, EssR, or a CenR/EssR mixture at various CenR:EssR molar ratios. Each phosphoryl transfer reaction was quenched with equal volume of SDS loading buffer. Reaction samples were loaded onto a 12% mini-PROTEAN precast gel (BioRad) and resolved at 180V at room temperature. The dye front of the gel was cut off and the rest of the gel was exposed to a phosphoscreen for 1–2h at room temperature. The phosphoscreen was imaged on a Typhoon phosphorimager (Cytiva), and gel band intensity was quantified using ImageQuant TL (Cytiva).

The identification of apoptotic cells by fluorescence microscopy and immunoblot analysis were described previously [16 (link)]. The protein bands were quantified using Image Quant TL software (Cytiva).

The nucleosomes (0.1 µM) or the 153-base-pair DNA (0.01 µM) and RAD51 or RAD51 mutants (0.24, 0.48 and 0.72 μM for DNA-binding assay, and 1.2, 2.4 and 3.6 μM for nucleosome-binding assay) were incubated at 37 °C for 30 min in the reaction buffer (20 mM HEPES-NaOH (pH 7.5), 15 mM NaCl, 1 mM MgCl₂, 1 mM dithiothreitol, 0.2 mM 2-mercaptoethanol, 0.03% NP-40 and 1.5% glycerol) in the absence or presence of 1 mM ATP, ADP or AMP-PNP. The samples were analysed by 4% non-denaturing polyacrylamide gel electrophoresis in 0.5× TBE buffer (45 mM Tris-borate and 1 mM EDTA), followed by ethidium bromide staining. Band intensities were quantitated by an Amersham Imager 680 with ImageQuant TL (Cytiva).

Lymphoblasts derived from a compound heterozygous DYT16 patient containing both P222L and C213R mutations as independent alleles were cultured alongside lymphoblasts derived from a family member containing no mutations in PACT as our control wt cells. Cells were plated at a concentration of 300,000 cells/ml of RPMI media containing 10% fetal bovine serum and penicillin/streptomycin. To analyze cellular response to ER stress, we treated cells with 5 μg/ml of tunicamycin (Santa Cruz) over a 24-h time course and harvested cells in RIPA (150 mM NaCl, 1.0% IGEPAL^® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) buffer containing a 1:100 dilution of protease inhibitor cocktail (Sigma) and phosphatase inhibitor (Sigma). Concentration of total protein extract was then determined using BCA assay and appropriate amounts of extracts were analyzed by western blot analyses using appropriate antibodies as indicated. When the cells were treated with luteolin prior to tunicamycin treatment, luteolin was added at 50 µM for 24 h. Quantification of band intensities was done using the Imagequant TL (Cytiva) software.

MEFs^Keap1−/− cells were lysed as previously described [68 (link)]. Cell lysates were separated by SDS-PAGE, and proteins were blotted to PVDF membranes. For detection, we used antibodies against the tags of the transfected proteins of interest in order to avoid the interference of endogenous proteins and the poor performance of commercially available antibodies for our purpose. Antibodies were used against Myc-Tag (#2276S, 1:1000 dilution) from Cell Signaling (Danvers, MA, USA), HA-Tag (#3724S, 1:1000 dilution) from Cell Signaling, beta-actin (#8457S, 1:1000 dilution) from Cell Signaling and FBXW11 (#13149-1-AP, 1:000 dilution) from Proteintech (Rosemont, IL, USA). Densitometric quantification was performed with ImageQuant™ TL, Cytiva (Tokyo, Japan)

For all densitometry measurements, we utilized ‘imagequant’ quant (ImageQuant TL, Cytiva) and statistical analysis and graphing was performed with Microsoft Excel software.

Selected CaMKIIβ isoforms were expressed in High Five insect cells via the baculovirus system. All purification steps were performed at 4°C. Cell pellets were resuspended in CaMKII lysis buffer (10 mM Tris–HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5% glycerol, and 1 mM DTT) supplemented with protease inhibitors (cOmplete; Roche) and lysed by sonication. Insoluble particles were separated by centrifugation at 21,500 rpm for 1 h (JA-25.50 fixed-angle rotor; Beckman Coulter). The soluble fraction was incubated with Strep-Tactin Sepharose beads (IBA Lifesciences) for 1 h and washed with CaMKII lysis buffer. Bound protein was eluted with CaMKII SEC buffer (50 mM Pipes, pH 7.5, 500 mM NaCl, 1 mM EGTA, 10% glycerol, and 1 mM DTT) containing 2.5 mM desthiobiotin (IBA Lifesciences). Eluted protein was concentrated and run on a Superose 6 10/300 Gl size-exclusion column (Cytiva) with CaMKII SEC buffer. Fractions were pooled according to SDS–PAGE and chromatogram, concentrated to ∼1 mg/ml, and flash-frozen in single-use aliquots in liquid nitrogen. Before use, aliquots were thawed on ice, gently mixed by pipetting, and centrifuged at 20,000 rcf for 5 min. Exactly equal concentrations were determined by repeated SDS–PAGE, Coomassie staining, and quantification with ImageQuant TL (Cytiva).