Effect of GRA on cellular kinase/phosphatase balance.
BMDM were infected with L. donovani promastigotes and treated with GRA (20 µM) for various time periods. (A) 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), PP2A (C) and SHP-1 (D) at mRNA and at protein level (E and F) 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 (G and H) and anti-p-p38 (I) 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. ***p<0.001; Student's t-test.