Effect of GRA on cellular kinase/phosphatase balance.

<p>BMDM were infected with <i>L. donovani</i> promastigotes and treated with GRA (20 µM) for various time periods. (<b>A</b>) Induction of PTP activity was measured using a PTP assay kit. Results are expressed as the relative increase (n-fold) over PTP activity in control cells. Expression of MKP1, MKP3 (<b>B</b>), PP2A (<b>C</b>) and SHP-1 (<b>D</b>) at mRNA and at protein level (<b>E and F</b>) were determined by Real time PCR and immunoblot analysis respectively. SHP-1, MKP1, MKP3 and PP2A were immunoprecipitated from infected and GRA (20 µM, 4 h)-treated macrophages and assayed for dephosphorylation activity using either recombinant p-ERK or p-p38 as substrate and visualizing by immunoblotting with anti-p-ERK (<b>G and H</b>) and anti-p-p38 (<b>I</b>) antibody. The amount of individual phosphatases was measured by stripping the blot and reprobing with antibodies against SHP-1, MKP1, MKP3 or PP2A. Results are representative of three individual experiments. Data represent the mean ± SD, n = 3. ***<i>p</i><0.001; Student's t-test.