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ads for 30 min at 4 C. Following a brief centrifugation, the supernatants had been removed and incubated with either agarose conjugated anti JAK2 antibody or anti NHE 1 antibody overnight at 4 C. Immunoprecipitates had been captured with 50 l of protein A G beads at 4 C for 1 hr. Then, the samples had been centrifuged and washed thrice with 1 ml (-)-MK 801 of RIPA buffer, and the proteins had been eluted from the beads utilizing 2x Laemmli sample buffer. Samples subsequently had been separated by SDS Page and transferred to PVDF membrane. Blots had been probed with anti calmodulin antibody , and, to ensure equal NHE 1 and Jak 2 precipitation from the samples, with NHE 1 monoclonal antibody or Jak 2 antiserum .
For phosphotyrosine immunoprecipitation experiments, quiescent podocytes grown onto 100 mm collagen coated tissue culture dishes had been pretreated with AG 490 , or with AG 1478 or vehicle for 30 min, then stimulated with 10 ng ml EGF or vehicle for 5 min and lysed in 0.5 ml 100 mm dish of RIPA buffer . Cell (-)-MK 801 lysates had been precleared by incubating with protein A agarose bead slurry for 30 min at 4 C. Precleared lysates had been incubated with monoclonal antiphosphotyrosine antibody conjugated to protein A agarose overnight at 4 C. The agarose beads had been collected by centrifugation, washed twice with RIPA buffer and as soon as with PBS, resuspended in 2x Laemmli sample buffer, boiled for 5 min, and subjected to SDS Page and subsequent immunoblot analyses with polyclonal antiphosphotyrosine, anti EGFR, anti Jak2, or with monoclonal anti CaM antibodies . Statistical Analysis Data had been analyzed by paired, two tailed Student’s t test and analysis of variance utilizing GraphPad Statistics Computer software.
P values 0.05 had been deemed BI-1356 significant. Results Immunohistochemical confirmation of podocyte differentiation Podocytes had been stained for WT 1 and synaptopodin. Undifferentiated podocytes did not stain for synaptopodin ; even so, the cells did stain for WT 1 . Differentiated podocytes stained for synaptopodin and WT 1 . The results of the staining confirm that in our hands, the cultured podocytes showed hallmarks of differentiation. EGFR mRNAs are expressed in podocytes Epidermal growth factor receptors constitute a family of four prototypical receptor tyrosine kinases . EGF receptor subunits dimerize upon ligand binding, resulting within the formation of activated receptors. We determined which EGFR subunit mRNAs had been expressed in podocytes utilizing RT PCR.
Undifferentiated podocytes expressed the HSP mRNAs for EGFR ErbB1, Neu HER2, ErbB3, and ErbB4 . Differentiated podocytes expressed the mRNAs for EGFR ErbB1, Erb3, and ErbB4. Neu HER2 mRNA was detectable at quite minute levels in differentiated podocytes . EGF induces concentration dependent increases in ECAR Getting established that podocytes express EGFR mRNAs, we next determined regardless of whether the cells expressed functional EGFR. We measured EGF induced increases in extracellular acidification rates utilizing microphysiometry below stop flow conditions. Figure 2B shows that EGF increased proton efflux inside a concentration dependent manner, confirming the presence of functional EGFR in differentiated podocytes. We next sought to establish the nature of the proton efflux pathway activated by EGF.
Because EGF has been shown to stimulate sodium proton exchangers in fibroblasts, esophageal epithelia and chondrocytes , we studied the expression of mRNAs encoding plasma membrane localized sodium proton exchangers NHE 1, NHE 2, NHE 3, and NHE 4. Figure 3A shows that differentiated podocytes express mRNA for NHE 1 and NHE BI-1356 2, with the levels of NHE 1 mRNA predominating. Undifferentiated (-)-MK 801 podocytes express only the mRNA for NHE 1 . The mRNAs for NHE 3 and NHE 4 had been not detected in undifferentiated or differentiated podocytes. Hence, it is achievable that EGFmediated proton efflux from differentiated podocytes entails NHE 1 or NHE 2.
To be able to test the involvement of sodium proton exchangers within the stimulation of proton efflux by EGF, we isotonically substituted tetramethylammonium for sodium within the BI-1356 extracellular perfusate, thereby removing the extracellular substrate for sodium proton exchangers. Figure 3B shows that EGF stimulated proton efflux inside a medium containing sodium, and that this effect was nearly abolished in medium in which sodium was replaced by TMA. Moreover, 5 M of 5 amiloride , an inhibitor of NHE 1 and NHE 2, attenuated EGF induced proton efflux by nearly 60 . These findings suggest that EGF induced increases in ECAR are resulting from NHE 1 or NHE 2 in podocytes. Calmodulin inhibitors, phosphotyrosine inhibitors and Jak2 inhibitors attenuate EGFinduced NHE 1 activity NHE 1 has two CaM binding domains which are crucial for its activation by many stimuli , whereas the function of CaM within the regulation of NHE 2 is considerably much less certain . Even though elevations of intracellular calcium increase the activity of NHE 2 , CaM has been shown to exert tonic inhibition on NHE 2 . To establish regardless of whether CaM is involved in EGF induced increases in ECAR, we analyzed