Here, we demonstrate that GSAP directly regulates -secretase activity and specificity. 6. ( 0.05, **** 0.0001; ns, not significant. To directly measure GSK1379725A the effect of GSAP on -secretase activity, we performed exo-cell assays (17) using recombinant APP or Notch substrate (18), which allows for the immediate and real-time analysis of -secretase activity for both substrates. HEK-APP WT and GSAP-KO cells were seeded in a 96-well plate overnight. The recombinant substrates were then added to the cells in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) and incubated for 2.5 h to measure -secretase cleavage. The cleaved products were detected with an AlphaLISA assay GSK1379725A (18). -Secretase activity was calculated by normalizing to protein concentration. HEK-APP GSK1379725A GSAP-KO cells have only 71% -secretase activity for A40 production compared with WT (Fig. 1and and and = 3. ( 0.05; ** 0.01; *** 0.001; ns, not significant. GSAP Modifies -Secretase Catalytic Efficiency for APP, but Not for Notch. To better understand the effect of GSAP on -secretase, we measured the kinetics of -secretase in membrane fractions prepared from four cell lines: HEK-APP GSAP WT and GSAP-KO cells transfected with EV or hGSAP. First, we found that and and and and and and GSK1379725A and em B /em ) -Secretase complex presented as transmembrane rods made up of PS1-NTF (green), PS1-CTF (red), Nct (purple), Aph1 (blue), and Pen2 (orange) in the presence ( em A /em ) of GSAP (blue sphere) in WT or in GSAP rescue GSK1379725A with induced PS1 conformation, which leads to -secretase activity for both APP and Notch. When GSAP is usually absent ( em B /em ) PS1 adopts a different conformation, which leads to a decrease in APP processing and a reduction in A secretion, but not in Notch processing. Materials and Methods Cell Culture. HEK-APP cell lines were cultured in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin. Human neuroblastoma SH-5YSY cell lines were produced in MEM/F-12 supplemented with 10% FBS and 1% penicillin. Transfection was done using Lipofectamine LTX with Plus Reagent according to manufacturers instructions. CRISPR-Cas9 GSAP-KO Generation and Isolation. Human GSAP CRISPR-Cas9 plasmid with gRNA targeting exon 16 (CATTGCCCTTTACAGTCATT) was design and cloned into PX459 by the Memorial Sloan Kettering Cancer Center (MSKCC) RNAi core facility. HEK-APP or SH-5YSY cells were transfected and selected with 2 g/mL puromycin. Single clones were isolated and analyzed by DNA sequencing of GSAP exon 16. Both HEK-APP and SH-5YSY hGSAP-KO clones contain a single-nucleotide deletion, which creates early termination. RNA Isolation and Real-Time RT-PCR. Total RNA was isolated with the QIAGEN RNeasy Mini Kit according to the manufacturers protocols. RNA (1 g) was reversely transcribed to cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen). qRT-PCR analysis was performed with designated cDNA samples using TaqMan Gene Expression Assay (Applied Rabbit polyclonal to APE1 Biosystems). All real-time qPCR was performed in triplicate on the Fast 7500 Real-Time PCR System (Applied Biosystems). TaqMan primers were hGSAP (Hs01383759_m1) and ribosomal 18S (Hs03003631_g1) from Applied Biosystems. Relative quantitation between samples was analyzed using the CT method. Meso Scale Discovery. Secreted human A species were detected using Meso Scale Discovery multiplex (6E10) from cell culture media 48 h posttransfection according to the manufacturers instructions. Western Blot and Antibodies. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH8.0, 150 nM NaCl, 0.1% vol/vol Nonidet P-40, and 0.5% wt/vol deoxycholic acid) containing protease inhibitor mixture. Protein concentration was determined by the DC Protein Assay Kit (Bio-Rad). Antibodies used for Western blot are as follows: PS1-NTF and Nct (from our laboratory), PS1-CTF (MAB5232; Millipore), Aph1a (38-3600; Invitrogen), Pen2 (18189; Abcam), APP (MABN10; Millipore), and HA (18181; Abcam). -Secretase Activity Assays. The exo-cell assay was performed as previously described (17). Briefly, cells were seeded in 96-well culture plates for 24 h and were washed with PBS after removing media. Next, Sb4 substrate (1 M) or NTM2 substrate (0.4 M) was added and incubated in 10 mM piperazine- em N /em , em N /em -bis(2-ethanesulfonic acid) (Pipes) buffer (50 mM Pipes,.