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Figure 7. CHEB42A produces a secreted peptide.A. Schematic of the construct used to study CHEB42A. CheB42a cDNA was tagged with GFP at the 5ā€² end and 3xHA tags at the 3ā€² end and was cloned into the mammalian expression vector pcDNA3.1 (Invitrogen). TM, transmembrane peptide as predicted by the SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/). predicted transmembrane domain. Predicted signal peptide cleavage site. T, N, and C represent the predicted full length and cleaved products. B. CHEB42A is a secreted protein. Cells were transfected with tagged cDNA (CheB42a) or pcDNA3.1 as a control. Proteins from cell lysates or cell media were immunoprecipitated (IP) with either anti-GFP (G) or anti-HA (H) antibodies, and then blotted for either tag. T, N, and C indicate the predicted peptides from Fig. 7A. C. Effects of CheB42a-conditioned media on ASIC1a currents. ASIC1a was expressed in Xenopus oocytes as in Fig. 6. Four ASIC1a-expressing oocytes were tested for pH-dependent currents with and without conditioned media from CheB42a-expressing cells. The same oocytes (Nā€Š=ā€Š4) were stimulated with unconditioned medium (pH 5.5), followed by stimulation with CheB42a-conditioned medium. There was no obvious effect of the conditioned media on pH-activation of ASIC1a channels. Currents were normalized in the conditioned state relative to the control media. All oocytes showed significant ASIC1a-dependent currents in response to pH 5.5, which does not elicit any currents in non-injected oocytes.

Image published in: Ben-Shahar Y et al. (2010)

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