HeLa cells were transfected with 25 or 50 nM pre-miRs (Applied Biosystems, USA), 2.5 ng/cm2; pRL control vector, and 50 ng/cm2; pGL3_HSV TK_3′UTR hNCSTN WT or C460T (rs1043329), T623G (rs113810300) or delCA515–516 (rs141849450) mutant variant plasmids. Twenty-four hours post-transfection, cells were lyzed, and luciferase activity was measured according to the manufacturer’s instructions (Promega, USA). For western blots, cells were lyzed in RIPA buffer [50 mM Tris HCl, 1% NP40, 0.9% NaCl, 0.25% Na-deoxycholate, 1 mM EDTA, 1× proteinase inhibitors (Roche, Basel, Switzerland), 1 mM PMSF, 1 mM Na3VO4, and 1 mM NaF], mixed with LDS sample buffer (Invitrogen, Carlsbad, CA, USA) containing 5% β-mercapto-ethanol and boiled at 95°C for 8 min. Crude extracts (10 μg) were immunoblotted with the Aph-1a [clone B80.2, kind gift from B. De Strooper (Nyabi et al., 2003 (link))], Pen2 (Cell Signaling, clone D2G6), Nicastrin (Cell Signaling, clone D38F9), Presenilin 1 (Millipore, clone PS1-loop), and Gapdh (Millipore, clone 6c5) antibodies. Membranes were detected using the ECL detection kit (Millipore, Billerica, MA, USA). Quantifications were performed using the Multi Gauge software (FUJIFILM, Minato-ku, Tokyo, Japan).
Nicastrin
Manufactured by Cell Signaling Technology
Sourced in United States
Nicastrin is a protein that plays a key role in the gamma-secretase complex. It functions as a scaffold, facilitating the assembly and activity of the gamma-secretase complex, which is involved in the processing of various transmembrane proteins.
Automatically generated - may contain errors
Lab products found in correlation
Presenilin 1, by Cell Signaling Technology (4 mentions)
β actin, by Merck Group (2 mentions)
Jagged 1, by Cell Signaling Technology (2 mentions)
Presenilin 2, by Cell Signaling Technology (2 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Pre mir s, by Thermo Fisher Scientific (1 mentions)
Ecl detection kit, by Merck Group (1 mentions)
Proteinase inhibitor, by Roche (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Merck Group (1 mentions)
Multi gauge software, by Fujifilm (1 mentions)
Presenilin 1, by Merck Group (1 mentions)
C myc, by Merck Group (1 mentions)
Nicastrin, by Merck Group (1 mentions)
Control rabbit igg, by Cell Signaling Technology (1 mentions)
Psd95, by Cell Signaling Technology (1 mentions)
Syvn1, by Santa Cruz Biotechnology (1 mentions)
Moab 2, by Biosensis (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
10 protocols using nicastrin
1
Luciferase Assay for Investigating 3'UTR Variants
2
Antibody Validation for Alzheimer's Research
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The mouse-specific membralin antibody used in this study was previously custom-made and now is available from Millipore (ABN1661, 1:1000). The PS1-NTF antibody used in this study was described previously (Rabbit polyclonal antibody, 1:1000)35 (link). Other antibodies were acquired from commercial sources as follows: c-Myc (Sigma, C2956, 1:1000), HA (Cell Signaling Technology, 3724 s, 1:1000), EMC3 (Santa Cruz, sc-365903, 1:1000), SYVN1 (Sigma, H7790, 1:500), SYVN1 (Santa Cruz biotechnology, sc-293484, 1:1000), AMFR (Cell Signaling Technology, 9590 s, 1:1000), AMFR (Santa Cruz Biotechnology, sc-166358, 1:1000), VCP (Thermo Fisher Scientific, MA3-004, 1:1000), VCP (Cell Signaling Technology, 2648 s, 1:1000), PS1-CTF (Millipore, 5232, 1:2000), human membralin (Sigma, HPA042669, 1:500), nicastrin (Cell Signaling Technology, 9447 s, 1:1000), nicastrin (Millipore, MAB5556, 1:1000), β-actin (Sigma, 5441, 1:10000), NICD (Cell Signaling Technology, 4147 s, 1:1000), PEN2 (Cell Signaling Technology, 8598 s, 1:1000), PSD95 (Cell Signaling Technology, 3450 s, 1:1000), active (cleaved) caspase3 (Cell Signaling Technology, 9662 s, 1:100), MOAB2 (Aβ40/42, Biosensis, M-1586-100, 1:500), rabbit IgG control (Cell Signaling Technology, 2729, 1 μg used as IP control).
3
Notch Signaling Pathway Regulation
4
Western Blot Analysis of Protein Expression
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Total proteins were extracted from cell lines and tissues with radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with a protease inhibitor cocktail (Calbiochem). Protein concentration was determined with the Pierce protein assay kit (Pierce). Total protein lysates (30 μg) were separated by SDS-PAGE, and the target proteins were specifically detected by western blotting with antibodies against RKIP (Santa Cruz Biotechnology, sc-28837), Notch1 (Cell Signaling Technology, 3608), cleaved Notch1 (NICD; Cell Signaling Technology, 4147), N-cadherin (Cell Signaling Technology, 4061), Snail (Santa Cruz Biotechnology, sc-28199), vimentin (Santa Cruz Biotechnology, sc-6601), Presenilin-1 (Cell Signaling Technology, 5643), Presenilin-2 (Cell Signaling Technology, 9979), PEN2 (Cell Signaling Technology, 8598), Nicastrin (Cell Signaling Technology, 9447), and β-actin (Sigma-Aldrich, A5441). Proteins were visualized with the Enhanced Chemiluminescence Detection Reagent (Pierce) and quantified using the Sigma-Gel software. Each data was corrected by b-actin and normalized to the control value.
5
Immunoblotting Analysis of Liver Proteins
6
CETSA Assay for CPX-Presenilin 1 Interaction
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The ability of CPX to interact with Presenilin 1 and Nicastrin was evaluated by using CETSA57 (link). Briefly, T24 cells were cultured and grown to 70–80% confluency. Cells were lysed using lysis buffer and diluted to the concentration of 3 mg/ml. The cell lysates were incubated with vehicle or CPX (20 μM) for 4 h. Next, the cell lysate was aliquoted into PCR tubes and subjected to heat gradient (44–80 °C) for 3 min. Subsequently, cells were centrifuged for 20 min. The resultant proteins were diluted with 4× Laemmli buffer, boiled at 95 °C for 5 min and loaded onto 10% SDS-PAGE gel, transferred to PVDF membrane and incubated overnight with Presenilin 1 (Cell Signaling # 5643) and Nicastrin (Cell Signaling # 3632) antibodies at a concentration of 1:1000. The western blot was visualized by Bio-Rad ChemiDoc-XRS+ instrument and analyzed by image lab software.
7
Alzheimer's Disease Protein Assays
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All chemicals were purchased from Fisher Scientific (Loughborough, Leicestershire, U.K.) unless otherwise stated. Actinomycin D was from Sigma–Aldrich (Gillingham, Dorset, U.K.).
Primary antibodies used: for APP (Y188; Abcam, Cambridge, U.K.; RRID:AB_2289606 and 22C11; Merck Millipore, Watford, Hertfordshire, U.K.; AB_94882), TDP-43 (Proteintech, Manchester, U.K.; AB_ 2200520), AICD (BioLegend, London, U.K.; AB_2564761, raised against a neo-epitope around the VMLK sequence at the ε-cleavage site), Tip60 (Novus Biologicals, Abingdon, Oxfordshire, U.K.; AB_1199339), Fe65 (Novus Biologicals; AB_789863), FLAG (Sigma–Aldrich; AB_439687), ADAM10 (Proteintech, Cat #25900-1-AP), BACE1 (Cell Signaling Technology, Leiden, The Netherlands; AB_1903900), nicastrin (Cell Signaling Technology; AB_10694688), Pen2 (ABcloncal, Woburn, MA, U.S.A.; AB_2771856), PS1 (Abcam; AB_1310605), PS2 (Cell Signaling Technology; AB_10831052), fibrillarin (Abcam; AB_1523617) and GAPDH (Proteintech, AB_2107436).
Primary antibodies used: for APP (Y188; Abcam, Cambridge, U.K.; RRID:AB_2289606 and 22C11; Merck Millipore, Watford, Hertfordshire, U.K.; AB_94882), TDP-43 (Proteintech, Manchester, U.K.; AB_ 2200520), AICD (BioLegend, London, U.K.; AB_2564761, raised against a neo-epitope around the VMLK sequence at the ε-cleavage site), Tip60 (Novus Biologicals, Abingdon, Oxfordshire, U.K.; AB_1199339), Fe65 (Novus Biologicals; AB_789863), FLAG (Sigma–Aldrich; AB_439687), ADAM10 (Proteintech, Cat #25900-1-AP), BACE1 (Cell Signaling Technology, Leiden, The Netherlands; AB_1903900), nicastrin (Cell Signaling Technology; AB_10694688), Pen2 (ABcloncal, Woburn, MA, U.S.A.; AB_2771856), PS1 (Abcam; AB_1310605), PS2 (Cell Signaling Technology; AB_10831052), fibrillarin (Abcam; AB_1523617) and GAPDH (Proteintech, AB_2107436).
8
Insulin Signaling in Adipocytes
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3T3-L1 cells (ATCC) were differentiated per standard protocol. Day 8–10 adipocytes were incubated with 200 nM Compound E overnight, serum starved for 4 h, then treated with 100 nM bovine insulin (Sigma) for 15 min prior to lysis. Both 3T3-L1 lysates and whole adipose extracts were lysed in Adipose Lysis Buffer (20 mM Tris, pH 7.4 150 mM NaCl, 10% glycerol, 2% Nonidet P-40, 1 mM EDTA, pH 8.0, 0.1% SDS, 0.5% sodium deoxycholate, 20 mM NaF, 30 mM NaPPi, 1 mM NaVO4), supplemented with Complete Protease Inhibitor Cocktail Tablet, EDTA-free (Roche). Immunoblots were probed with antibodies against Nicastrin (#5665), Psen2 (#2192), phospho-Akt Thr308 (#9275), total Akt (#9272), phospho-GSK-3β Ser9 (#9322), total GSK-3β (#9315), tubulin (#2148), and actin (#8456) from Cell Signaling.
9
Notch Signaling Pathway Analysis
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Antibodies against Notch1, Notch2, Jagged1, Hes1, Nicastrin, Dll4, E‐Cadherin, N‐cadherin, Slug, Snail and β‐actin were obtained from Cell Signaling Technology (Danvers, MA). All other chemicals were purchased from Fisher Scientific (Suwanee, GA) and Sigma‐Aldrich (St. Louis, MO).
10
Western Blot Analysis of Cellular Proteins
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Cells were lysed with lysis buffer containing 50 mM Tris (pH 7.4), 140 mM NaCl, 5 mM EDTA, and a protease inhibitor tablet, followed by sonication. Lysate protein concentrations were determined using Bradford reagents (AMERSCO, MA, USA). Proteins were resolved by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes (Pall Corporation, New York, NY, USA), and incubated with blocking buffer (5% nonfat dry milk in TBS and 0.1% Tween 20) for 30 min. Western blot analyses were performed using primary antibodies against NF-κB, NLRP3, ASC, nicastrin, pen2 (Cell Signaling Technology, Danvers, MA, USA), Aph1 (Invitrogen), amyloid C-terminal fragment (Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (Bethyl Laboratories, Mongomery, TX, USA). Secondary antibodies of rabbit (Promega), rat, and goat (Bethyl Laboratories) were used, and detection was performed with SUPEX ECL reagent (Neuronex, Korea) and an ImageQuant LAS-4000 (GE Healthcare, MA, USA), according to the manufacturer’s instructions. The integrated blot density was quantified through ImageJ.
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