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. Author manuscript; available in PMC: 2008 Aug 18.
Published in final edited form as: J Proteome Res. 2006 Jul;5(7):1636–1646. doi: 10.1021/pr0502469

Figure 5. Phosphorylation of Hsp70, Hsp90, Grp78, and PDI in Mesangial Cells.

Figure 5

Mesangial cells were treated with the PI-3K inhibitor LY294002 (25μM) or vehicle for 1 hr, prior to stimulation with PDGF. Lysate were prepared in urea/thiourea buffer and exchanged to 2 M urea kinase buffer. Separate aliquots of PDGF-stimulated lysate were incubated with 0.5 Units protein phosphatase 2-A (PP2A) for 1.5 hr at 30°C. Proteins were separated by 2-DE using IPG strips pH 4−7 (Hsp90, Hsp70, and Grp78; panel A, B, and C) or pH 4.5−5.5 (PDI; panelD), and specific proteins identified by immunoblot analysis. Panel A, 30 min of PDGF stimulation increased abundance of more acidic Hsp90 species. Both LY294002 and phosphatase treatment reversed the PDGF-induced acidic shift in Hsp90. Panel B, PDGF induced an acidic shift in Hsp70 by 10 min of stimulation and this effect was diminished by 30 min. Treatment with LY294002 and PP2A prevented and reversed, respectively, the acidic shift in Hsp70 pI induced by PDGF. Panels C & D, 30 min of PDGF stimulation increased abundance of the most acidic species of Grp 78 and PDI and this effect was inhibited by pre-treatment with LY294002.