Anti rcas1

Anti-ALDH2 (Proteintech, 15310-1-AP, 1:4000), anti-4-HNE (Abcam, ab46545, 1:2000), anti-APP C-Terminal Fragment (C1/6.1, Biolegend, 802801, 1:500), anti-β-amyloid (1–16) (6E10, Biolegend, 803001, 1:1000), anti-β-amyloid (1–40) (Cell Signaling Technology, 12990, 1:1000), anti-β-amyloid (1–42) (Cell Signaling Technology, 14974, 1:1000), anti-GM130 (Cell Signaling Technology, 12480S, 1:1000), anti-TGN46 (Invitrogen, MA3-063, 1:1000), anti-presenilin 1 (Sigma, MAB5232, 1:500), anti-PEN2 (Abcam, ab18189, 1:500), anti-APH1 (Invitrogen, PA1-2010, 1:1000), anti-Rab5 (Cell Signaling Technology, 3547, 1:1000), anti-RCAS1 (Cell Signaling Technology, 12290S, 1:1000), anti-VDAC (Cell Signaling Technology, 4661S, 1:1000), anti-LAMP2 (Proteintech, 27823-1-AP, 1:1000), anti-CANX (Cell Signaling Technology, 2679, 1:1000), anti-VPS35 (Abcam, ab10099, 1:1000), anti-LC3A (Cell Signaling Technology, 4599, 1:1000), anti-Iba1 (Wako, 019-19741, 1:500), anti-tau (Cell Signaling Technology, 46687, 1:1000), anti-phospho-S396-tau (Abcam, ab32057, 1:1000), anti-α-tubulin (GeneTex, GTX628802, 1:10,000), anti-β-actin (GeneTex, GTX124213, 1:10,000), anti-GAPDH (MBL, M171-3, 1:10,000), and anti-SorLA/SORL1 antibody (Abcam, ab190684, 1:1000).

RPE cells were seeded on cover glasses 1 s (Matsunami; Osaka, Japan) 24 h prior to the experiment to obtain cells in the exponential growth phase. Cells in G2 phase were identified by CENPF staining. To identify cells in S phase, EdU was added 30 min prior to X-ray irradiation. Next, cells were fixed for 10 min in 3% paraformaldehyde–2% sucrose and permeabilized for 3 min with 0.2% TritonX-100-phosphate buffered saline (PBS). Cells were washed twice with PBS and incubated at 37 °C for 30 min with the primary antibody in Solution A (TOYOBO; Osaka, Japan). Cells were then washed with PBS and incubated at 37 °C for 30 min with secondary antibodies conjugated to Alexa Fluor 488/594 in 2% bovine serum albumin (Sigma-Aldrich)/PBS containing 0.1 mg/mL 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI; Roche, Mannheim, Germany). Subsequently, cells were stained with Click-iT™ EdU. After an additional wash with PBS, cover glasses were mounted in Vectashield (Vector Laboratories; Burlingame, CA, USA). The following primary antibodies were used: anti-RCAS1 (1:400, #12290; Cell Signaling Technology, Danvers, MA, USA) and anti-CENPF (1:400, #610768; BD Biosciences, Franklin Lakes, NJ, USA). The following kit was used for EdU staining: Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 647 dye (Thermo Fisher Scientific; Waltham, MA, USA).

The anti-FLAG and anti-caspase-11 antibodies were from Sigma. The anti-SREBP1, anti-HMGCS1, anti-HMGCR antibodies were from Santa Cruz Biotechnology. The anti-HA antibody was from Roche, and the anti-S1P and anti-GAPDH antibodies were from Abcam. The anti-RCAS1, anti-EEA1, anti-LAMP1, anti-human caspase-4 and anti-caspase-5 antibodies were from Cell Signaling Technology. The LPS was purchased from Sigma (L2637).