To determine whether FABP4 have any effect on eNOS activity, we studied the eNOS activation measuring its phosphorylation at Ser.
To rule out other possibility of MnSOD induction by BTG2 expression, the activity of FOXO3a, a well known transcription factor of MnSOD in quiescent cells against oxidative stress [ 33], was analyzed by measuring its phosphorylation on serine253 residue, however, there was no significant difference of its activity after BTG2 expression (Additional file 1: Figure S1A).
Previous studies have focused on monitoring TRPM7 kinase activity by measuring its autophosphorylation and phosphorylation of the exogenous substrate MBP [ 2, 3, 26].
The kinase activity of the purified recombinant AtWAKL10431 700 protein was assessed in vitro by measuring its ability to direct the phosphorylation of a special Ser/Thr substrate peptide as described in the Omnia™Ser/Thr-Recombinant Kit (KNZ2011, BioSource, Nivelles, Belgium).
PLX4720 did not appreciably affect the activity of GSK3α/3β, as measured by its phosphorylation at Ser21 or Ser9, respectively26, nor the pathways associated with Akt activation, as indicated by Ser473 and Thr308 phoSupplementary7 (Supplementary Fig. 2h).
Rad53 activation was measured by its phosphorylation-dependent shift in electrophoretic mobility.
Exemplified by the total mammalian target of rapamycin (mTOR), a master regulator of protein synthesis, they found a slight decrease in its protein content, unfortunately with no measures of its phosphorylation status or activity [11].
This analysis showed that applications of the two clinically relevant CML drugs, IM (1 μM) and DST (0.01 and 1 μM), provoke distinct alterations in the electrophoretic mobility of Gab2, a rough measure of its phosphorylation status.
The activity of mTOR can be assayed by measuring the phosphorylation its downstream targets, such as p70S6 kinase or S6 ribosomal protein (1).
Mammalian target of rapamycin (mTOR) inhibits autophagy and its kinase activity can be detected by measuring the phosphorylation of its substrates, such as p70S6 kinase (S6K1).
For that purpose, we developed a modified version of the previously described KIRA-ELISA [28], a rapid, sensitive and high-capacity assay that quantifies TrkB activation in ELISA microtiter plates by measuring phosphorylation of its tyrosine residues [see Data S1 and Fig. S1,S2].
