Sc 47724

Protein lysates were extracted in ice-cold lysis buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EGTA, 25 mM NaCl, 1% Nonidet P-40, 1 mM Na3VO4, 10 mM NaF, 0.2 mM leupeptin, 1 mM benzamidine, and 0.1 mM 4⁻(2-aminoethyl)-benzenesulfonylfluoride hydrochloride). Protein content was determined using BCA Protein Assay kit (Pierce). Proteins were subjected to SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with antibodies, ZNF521 (1:750) Sigma–Aldrich, SAB3500840; GAPDH (1:3000) Santa Cruz sc-47724; P16^INK4 (1:500) Abcam 201980; mTOR (1:500) Cell Signaling 4517; Rb (1:500) Santa Cruz sc-50; p53 (1:500) Cell Signaling 2524; p21^CIP1 (1:500) Santa Cruz sc-6246; BMP4 (1:1000) Abcam 39973; β-tubulin (1:2500) Cell Signaling 2128; ZNF423 (1:500) Santa Cruz sc-48785; Lamin A/C (1:500) Santa Cruz sc-6215; TNFα (1:500) Cell Signaling 6945; Horse anti-mouse IgG, (1:2000) Cell signaling 7076; Goat anti-rabbit IgG (1:2000) Cell signaling 7074; Donkey anti-goat IgG (1:1000) Santa Cruz sc-2020. A summary of antibodies used is found in Supplementary Table 4. For development ChemiDoc Imaging System (Bio-Rad) was used. Uncropped and unprocessed scans with ladders are shown in the Source Data file.

The expression level of LRP1 and APP were measured by Western blot. Briefly, protein extracts were obtained from whole cell lysates and quantified with the Pierce BCA assay from ThermoFisher Scientific (Waltham, MA). For SDS-PAGE 10% gels were used, 10 μg of protein sample was loaded and electrophoresis was run at 150 mV for 70 min. Protein transfer to a previously methanol-activated PVDF membrane was carried out at 400 mA for 1 h at 4 °C. Membranes were blocked in blocking buffer (5% milk in TBS-Tween20) for 1 h at room temperature with shaking. Overnight incubation with primary antibodies in blocking buffer was done at 4 °C with shaking. After four 5-min TBS-Tween20 washes, membranes were incubated with secondary antibodies in blocking buffer for 4 h at room temperature with shaking. After another round of four 5-min TBS-Tween20 washes, membranes were developed with X-ray films and ECL reagent from Santa Cruz Biotechnology (Heidelberg, Germany). The antibodies and dilutions used were as follows: LRP1 1:20,000 (Abcam #ab92544) (Cambridge, UK), APP 1:1,000 (Abcam #ab15272), GAPDH 1:2,000 (Santa Cruz #sc-47724), Beta-actin 1:5,000 (Abcam #ab8227), HRP-secondary goat anti-rabbit 1:5,000 (Abcam #ab6721), HRP-secondary goat anti-mouse 1:5,000 (Dako-Agilent Technologies #P0447) (Santa Clara, CA).