| Assay Method Information | |
| | Cell-Based Assay for Detection of SMYD2 Methylation Activity |
| Description: | For the detection of SMYD2 cellular methylation activity an In Cell Western (ICW) assay was established. This assay allows rapid processing of multiple samples for SMYD2 methylation derived immunofluorescence signals, with normalization to cell number via the use of the nucleic acid dye DRAQ5. KYSE-150 cells (human esophageal carcinoma cell line; DSMZ-German Collection of Microorganisms and Cell Cultures; No: ACC 375) have been stably transfected with a construct expressing wild-type SMYD2 (NCBI Reference Sequence: NP_064582.2). To detect SMYD2-mediated methylation signals in cells, a customized antibody directed against mono-methylated lysine 370 on protein p53 (p53K370me1) was used. The polyclonal antibody was generated (Eurogentec) against a p53 peptide containing the mono-methylated K370 epitope as described elsewhere (Huang et al., Nature, 2006, 444(7119):629-32).For conducting the ICW assay 5000 SMYD2 overexpressing KYSE-150 cells/well were seeded in 96-well plates (SIGMA) and cultivated for 24 h. As a control for maximal inhibition of ectopic methylation activity, non-transfected KYSE-150 cells were used. Cells were grown in 49% RPMI 1640 with 49% Ham's F12 media supplemented with 2% heat inactivated fetal calf serum (FCS). For determination of SMYD2 inhibitory activity, cells were treated for 72 h in the presence of compounds or with DMSO. Cells were treated with compounds to be tested at a final concentration range varying from 3.9×10−8 to 5×10−6 M. Media was removed and 3.7% (w/v) formaldehyde in PBS was added for 20 min. After two washes with phosphate buffered saline (PBS), 0.25% (v/v) Triton X100 in PBS was added for 15 minutes of permeabilization. After one washing step with PBS, cells were blocked for 1 h with 5% (w/v) non-fat dry milk in PBS. Fixed cells were exposed to primary p53K270me1 antibody in 5% non-fat dry milk in PBS for 24 h. One row of cells on each plate was not exposed to p53K370me1 antibody and was reserved for background control measurements. The wells were washed three times with PBS, then secondary IR800 conjugated antibody (LI-COR) and DNA-intercalating dye, 5 μM DRAQ5 (LI-COR) were added for 3 h. After 5 washes with PBS, the fluorescence in each well was measured on an Odyssey (LI-COR) scanner at 800 nm (p53K370me1 signal; 764 nm excitation) and 700 nm (DRAQ5 signal; 683 nm excitation). Fluorescence intensity was quantified and normalized to background and DRAQ5 signals. IC50 values were calculated by fitting the normalized inhibition data to a 4-parameter logistic equation (Minimum, Maximum, IC50, Hill; Y=Max+(Min−Max)/(1+(X/IC50)Hill)) for each tested compound. For IC50 determination C0 (=no inhibition) was defined as the signal measured for DMSO treated controls. Ci (maximal inhibition) was defined as the signal measured for non SMYD2 overexpressing KYSE150 cells. |
| Affinity data for this assay | |
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