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Effect of GRA on cellular kinase/phosphatase balance.

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posted on 20.02.2013, 10:49 by Anindita Ukil, Susanta Kar, Supriya Srivastav, Kuntal Ghosh, Pijush K. Das

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.