Ab108208

Equal amounts of total protein (15 or 20 µg) were fractionated by SDS‐PAGE under reducing conditions (4%‐12% TGX gels, Bio‐Rad, Marnes la Coquette, France) and blotted onto nitrocellulose membranes (Bio‐Rad). The membranes were blocked with 5% BSA in TBS containing 0.1% Tween 20 (TBS‐T) and hybridized with the primary antibody of interest overnight at 4°C. Membranes were washed in TBS‐T and then hybridized with the secondary antibody for 1 hour at room temperature. Antibodies were diluted in TBS‐T containing 5% BSA. The membranes were washed with TBS‐T, and immune complexes were revealed by enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific) and imaged using the LAS‐3000 Luminescent Image analyzer and Image Gauge software (Fuji, FSVT). The antibodies used for Western blotting were directed against beta‐actin (Sigma‐Aldrich, #A2668), cleaved caspase 7 and 8 (Cell Signaling Technology, #9491 and “9496, respectively), LRP8 and cleaved PARP (Abcam, #ab108208 and #ab108208, respectively).

Brains were dissected and immediately frozen in liquid nitrogen. Proteins were obtained from frozen tissue by homogenizing tissue in a modified RIPA buffer (Jodelka et al, 2010) using a power homogenizer. Cells were lysed with Laemmli buffer. Proteins were boiled in sample buffer and separated on 8% SDS–PAGE and then transferred to PVDF membranes (Millipore). Membranes were blocked in 5% nonfat dry milk in 1× TBST and incubated with ApoER2 C‐terminal antibody ab108208 (1:2,000) (Abcam), ApoER2 exon 19‐specific antibody (1:1,000) (a gift from Joachim Herz) (Beffert et al, 2005), GFAP antibody (1:5,000) (ab7260, Abcam), SRSF1‐specific antibody (1:100)(a gift from Adrian Krainer), MetAP2‐specific antibody (1:200) (D3I1H, Cell Signalling), or β‐actin‐specific antibody (1:2,000) (Sigma‐Aldrich) followed by incubation with anti‐rabbit or anti‐mouse HRP‐conjugated antibody (1:5,000) (Thermo). Bands were visualized using ECL reagents (Millipore) and by exposing on autoradiography film. Blots were quantified using Image J software v1.48s (NIH).

Protein extracts were separated by 1D gel electrophoresis using NuPAGE 4–12% (w/v) Bis-Tris midi gels (Invitrogen, USA), and transferred onto PVDF membranes using an iBlot 2 transfer system (Invitrogen, USA), according to the manufacturer’s specifications. Immunoblots were performed to detect FLAG tag (1:1000, D6W5B, Cell Signaling Technology, USA), LRP8/APOER2 (1:2000; ab108208, Abcam, UK), β-ACTIN (1:5000, AC-15, Sigma-Aldrich, Australia), GPX4 (1:1000, ab125066, Abcam, USA), Notch1 (1:1000, D1E11, Cell Signaling Technology, USA), PS1 (1:1000, D39D1, Cell Signaling Technology, USA), or PS2 (1:1000, D30G3, Cell Signaling Technology, USA), SELENOP (B-9, Mouse mAb; sc-376858, Santa Cruz Biotechnology, Inc., USA), cleaved caspase 3 (Asp175; 1:1000, 5A1E, Rabbit mAb 9664; Cell Signaling Technology, USA), or cleaved caspase 7 (Asp198; 1:1000, D6H1, Rabbit mAb 8438) followed by appropriate HRP-conjugated secondary antibodies (1:5000, Thermo, Australia). Chemiluminescence was detected using Pierce ECL (Thermo, Australia) and visualized using the LAS-3000 Imaging System (Fujifilm, Japan) or Odyssey® Fc Imaging System (LI-COR Biosciences, Lincoln, NE). Representative blots for all proteins (including β-actin) are shown. Original western blots for all relevant figures are shown in “Supplementary Material—Original Blots”.