Antisense and siRNA downregulation of STAT3 abrogated EGFR-mediated transcription signalling as evinced by inhibition of electrophoretic mobility shift assay (EMSA) shift detection.
This modification causes an electrophoretic mobility shift allowing convenient detection of in vivo ASK activity by western blot analysis [ 20, 24- 26, 54]. A. thaliana seedlings expressing BZR1-CFP were treated with different concentrations of compound 10 and 15 and the BZR1 phosphorylation pattern was compared by western blot analysis with that of mock treated plants.
The increased sensitivity of the concentration-enhanced mobility shift platform allows for detection of kinase activities from single cell lysate even upon dilution into a much larger reaction chamber.
Previous reports have established that this platform is capable of achieving very high preconcentration factors (million fold), and has been successfully used in many biological assays including enhancing protein binding kinetics, improving sensitivity of enzyme assays, and reducing the limit of detection of mobility shift assays.
The molecular weight of 65 kDa as compared to the predicted 59.4 kDa suggested the presence of glycosyl modifications, as demonstrated using a glycosylation detection reagent and a mobility shift of about 5 kDa after PNGase F treatment (data not shown).
Mobility shift of the probe was detected due to the formation of one distinct complex.
Electrokinetic preconcentration coupled with mobility shift assays can give rise to very high detection sensitivities.
Phosphorylation was detected as a mobility shift on a polyacrylamide denaturing gel.
As the intracellular contents of a single cell (pL volume) is then diluted into a nanoliter sized chamber, the ultrasensitive assay provided by the concentration-enhanced mobility shift assay platform is an enabling technology that allows detection of this low-abundance kinase activity.
In vitro sumoylation assay, mass spectrometry detection of sumoylated lysines, and electrophoretic mobility shift assays (EMSAs) were performed as described previously, except that the EMSA used a 5% polyacrylamide gel in 0.5 × Tris-borate-EDTA and 6 nM DNA substrate (Sarangi et al., 2014).
