Sc 8354

Reagents were purchased from Sigma-Aldrich unless specified otherwise. Antibodies used in this study were as follow: anti-APP 6E10 (SIG-39320, Covance), anti-Aβ42 12F4 (SIG-39142, Covance), mouse anti-Tau Tau12 (SIG-39416, Covance), mouse anti-Tau HT7 (MN1000, Thermo Scientific Pierce), mouse anti-phosphorylated Tau AT8 (MN1020, Thermo Scientific Pierce), rabbit anti-Tau H150 (SC-5587, Santa Cruz Biotechnology), rabbit anti-Tau (A0024, Dako), rabbit anti-clusterin H330 (SC-8354, Santa Cruz Biotechnology), goat anti-clusterin M18 (SC-6420, Santa Cruz Biotechnology), goat anti-clusterin C18 (SC-6419, Santa Cruz Biotechnology), mouse anti-BIN1 99D (05-449, Millipore), rabbit anti-Gapdh (G9545), rabbit anti-actin (A2066), and mouse anti-Flag (F1804), mouse anti-myc 9E10 (SC-40, Santa Cruz Biotechnology), mouse and goat IgG control (SC-2015 and SC-2028, Santa Cruz Biotechnology), donkey anti-mouse IgG (H+L) IRDye 800 (926-32212, LI-COR Biosciences), donkey anti-rabbit IgG (H+L) IRDye 680 (926-68073, LI-COR Biosciences), donkey anti-rabbit IgG (H+L) IRDye 800 (926-32213, LI-COR Biosciences), and donkey anti-goat IgG (H+L) IRDye 680 (926-68074, LI-COR Biosciences).

The proteins were prepared by suspending the cells in lysis buffer with complete ethylenediaminetetraacetic acid (EDTA)-free protease-inhibitor-cocktail tablets (Roche, Basel, Switzerland) for 10 min on ice. The cell extracts were sonicated (3x10 sec), then centrifuged at 15,000 × g for 15 min to remove the insoluble materials.
Each protein sample was electrophoresed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA). After blocking with 5% fat-free milk powder at room temperature for 2 h, the membranes were incubated with primary antibodies against CLU (sc-8354), Nestin (sc-33677), Musashi RNA-binding protein 1 (Msi-1; sc-135721), MAP-2 (sc-74421), GFAP (sc-33673), MBP (sc-13564) and β-actin (sc-8432; Santa Cruz Biotechnology, Inc.) at 4°C overnight, and subsequently cultured with HRP-conjugated goat anti-mouse IgG (sc-2489; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. β-actin was used as an internal control. The reaction product was visualized by enhanced chemiluminescence (ECL; Amersham Pharmacia, Piscataway, NJ, USA).

Proteins were extracted from the cell layer and 5–20 µg of total protein subjected to non-reducing SDS-PAGE using 12.5% resolving/ 4.8% stacking polyacrylamide gels. Electrophoresed proteins were electroblotted onto polyvinyldene difluoride (PVDF) membrane and immunodetection was carried out in Tris-buffered saline Tween-20 pH 7.6 (10 nM Tris, 150 nM NaCl/0.1% v/v Tween 20) with 5% w/v non-fat milk. Polyclonal rabbit or mouse-monoclonal anti-human clusterin (0.4 µg/ml, sc-8354 (discontinued) or sc-5289, Santa Cruz) or mouse monoclonal anti-human α-smooth muscle actin (7.1 ng/ml, M0851, Dako, Denmark) and goat polyclonal anti-human vinculin (0.4 µg/ml, sc-7649, Santa Cruz) antibodies were incubated overnight at RT. Secondary antibodies conjugated to horseradish peroxidase (HRP) (goat anti-rabbit, 50 ng/ml; rabbit anti-mouse, 260 ng/ml; rabbit anti-goat, 110 ng/ml, Dako, Denmark) were applied for 1.5 hours at RT. Signal detection via chemiluminescence (Luminata Crescendo Western HRP substrate, Millipore) was captured via ImageQuant^TM and acquisition tool ImageQuant TL 1D v.8.1 analysis software (GE healthcare, UK). Settings: Exposure type – “Precision”, exposure time −10 seconds, high sensitivity/ resolution. Protein sizes were analyzed via PageRuler Pre-stained Protein Ladder (Thermo Fisher Scientific, UK).