Figure 3.

Activation of PKC stimulates SorL1 ectodomain shedding and SorL1 phosphorylation. Wt MEFs were grown to confluency and incubated for 10 min in the presence of vehicle (-), 1 uM PDBu and/or 1 uM Okadaic acid (OA) (+). Equal volumes of media and protein lysate were subjected to SDS PAGE, transferred to PVDF and probed with α-SorL1 (N-terminal BD Biosciences) to detect holo SorL1 and the soluble SorL1 (sSorL1) shed ectodomain, or α-SorL1 C-terminus (J. Lah, Emory) to detect SorL1 CTFs and α-actin as a loading control. A. Treatment with 1 μM PDBu (PKC activator) alone and in combination with 1 uM Okadaic acid, increased ectodomain shedding of SorL1 (sSorL1) and sAPPα into the media and increased SorL1 cell associated CTF generation. The migration of the SorL1 CTF appeared slightly retarded upon stimulation of PKC and inhibition of PP1 and PP2A. B. Treatment of lysates with 1 μM PDBu increased levels of the largest molecular weight immunopositive SorL1 species.