680lt goat anti mouse igg

LT protein turnover was measured by a cycloheximide (CHX) chase assay using quantitative immunoblot analysis. LT plasmids were co-transfected with an eGFP plasmid to normalize transfection efficiency, and all experiments were performed in triplicate. Cells were lysed in IP buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% TritonX-100, 1 mM PMSF, 1 mM benzamidine) and whole cell lysates without pre-clearing were used for direct immunoblotting. Primary antibodies were incubated overnight at 4 °C, followed by 1 h of secondary antibody incubation at room temperature. All signals were detected using quantitative infrared (IR) secondary antibodies (IRDye 800CW goat anti-mouse, 800CW goat anti-rabbit, 680LT goat anti-rabbit IgG, 680LT goat anti-mouse IgG) (LI-COR); signal intensities were analyzed using a laser-scanning imaging system, Odyssey CLX (LI-COR). CM2B4 (Santa Cruz Biotechnology, Dallas, TX, USA), 2T2 (Millipore, Billerica, MA, USA), PAb416 (Santa Cruz Biotechnology), c-Myc (9E10, Santa Cruz Biotechnology), GFP (D5.1, Cell Signaling) HA-Tag (C29F4, Cell Signaling, Danvers, MA, USA), anti-FLAG (M2, Sigma-Aldrich, St. Louis, MO, USA), anti-α-Tubulin (12G10, DSHB), β-Actin (13E5, Cell Signaling) antibodies were used for this study.

Stably transduced BJ-hTERT cells (selected with puromycin for 14 days) were lysed with an IP buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% TritonX-100, 1 mM PMSF, 1 mM benzamidine) and whole cell lysates were used for immunoblot analyses. Membranes were incubated with a primary antibody overnight at 4 °C, and then incubated with a secondary antibody for 2 h (h) at room temperature (RT). A quantitative infrared (IR) imaging system, Odyssey CLX (LI-COR), was used to determine protein expression. The primary antibodies used in this study included phospho-p53 (Ser15) (Cell Signaling, 9284, Danvers, MA, USA), p21 Waf1/Cip1 (Cell Signaling, 2947), cyclin E (Santa Cruz Biotechnology, sc-247, Dallas, TX, USA), phospho-histone H3 (Ser10) (Cell Signaling, 53348), GFP (Santa Cruz Biotechnology, sc-9996), and β-Actin (Cell Signaling, 4970). All signals were detected using quantitative infrared (IR) secondary antibodies (IRDye 800CW goat anti-mouse, 800CW goat anti-rabbit, 680LT goat anti-rabbit IgG, 680LT goat anti- mouse IgG) (LI-COR).

Synthetic human Aβ_(1-42) (H-1368) was obtained from Bachem Distribution Services GmbH (Bubendorf, Germany), and Aβ_(1-42) oligomers were prepared according to the original protocol of Klein's group [15 (link)]. Aβ_(1-42) monomers were prepared from HIFP-treated Aβ_(1-42) according to our previously published protocol [16 (link)]. For dot blot analysis, protein samples (0.5 μg of each) were spotted onto a nitrocellulose membrane. The membrane was first probed with the rabbit polyclonal anti-oligomer A11 antibody (Thermo Fisher Scientific, 1:100 – Waltham, MA, USA), and then re-probed with the mouse monoclonal antibody 6E10 (BioLegend, 1:800 – San Diego, CA, USA), which detects all forms of Aβ (Supplementary Fig. S2A). For western blot analysis, protein samples (7.5 μg of each) were separated by 4-12% Bis·Tris SDS-PAGE and transferred to a nitrocellulose membrane. Then, the membrane was blotted with the mouse monoclonal antibody 6E10 (Supplementary Fig. S2B). Membranes were incubated with IRDye^® 800CW goat anti-rabbit IgG or 680LT goat anti-mouse IgG secondary antibodies (1:15,000, LI-COR 926-32211 and 926-68020, respectively) for 1 hr at room temperature (RT). Hybridization signals were detected with the Odyssey^® Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).