Figure 4.

Loss of PAFAH1B2 in HEK293–APPsw and primary MEF cells reduces APP CTFs. A, HEK293–APPsw cells virally transduced with shRNAs against PAFAH1B2 resulted in highly potent knockdowns of PAFAH1B2 compared with nontargeting control. This correlated with a dramatic reduction of both the αCTF and βCTF. A shorter exposure of the immunoblot showed that full-length mature (m.) and immature (im.) APP levels were unaffected. Actin was also unaffected. B, In primary MEF cells, analysis of APP and APLP2 processing by Tris–tricine SDS-PAGE revealed that, in the absence of PAFAH1B2, endogenous APP and APLP2 CTFs were dramatically reduced. No such change was observed for MEFs lacking PAFAH1B3. As before, the level of full-length APP was slightly elevated in PAFAH1B2^−/− MEFs. C, Analysis of N-cadherin (NCAD) and E-cadherin (ECAD) processing by 10% SDS-PAGE revealed full-length proteins and CTFs running at ∼45 and 36 kDa, respectively. For E-cadherin, the level of full-length protein was slightly increased in the absence of PAFAH1B2, but for both E- and N-cadherin, the CTF levels were unchanged in the absence of either PAFAH1B2 or PAFAH1B3. D, HEK293–APPsw/F-NEXT cells virally transduced with shRNAs against PAFAH1B2 resulted in potent knockdowns of PAFAH1B2 compared with nontargeting control. In contrast to the reduced levels of APP CTFs, the levels of F-NEXT and its γ-secretase generated product, NICD, were completely unaffected. MW, Molecular weight.