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. 2010 Jun 23;24(8):1543–1558. doi: 10.1210/me.2009-0381

Figure 4.

Figure 4

Recombinant PAM-CD localizes to the nucleus. A, Cleavage of PAM-1 in exon 16 occurs in LDCVs, generating membrane-anchored PAL (PALm); cleavage after PAL produces 22-kDa TMD-CD (8). Intramembrane proteolysis is thought to create soluble, cytosolic sf-CD (16 kDa) (8). The structures of mycTMD-CD and mycCD, which were stably expressed in AtT-20 cells, are shown for comparison. B, Alexa647 labeled PAM-exon16 or PAM-CD was microinjected into AtT-20 cells or neurons; white arrow marks the injection site. Representative confocal images taken at the times indicated are shown along with a differential interference contrast (DIC) image. Scale bars, 10 μm. C, AtT-20 cells or neurons were microinjected with free Alexa647 dye, Alexa647-Exon16, Alexa647-PAM-CD, or Alexa647-PAM-CD-5P (with five phosphomimetic mutations). Images were taken no longer than 2 min after injection. Scale bar, 10 μm. D, Quantification of the nuclear/cytoplasmic ratio of images taken as in panel C; mean ± sem. The number of cells analyzed is indicated. a.u., Arbitrary units.