D6w5b

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”.

Anti-HA (rabbit monoclonal C29F4, 1:1000, Cell Signaling Technology); anti-ataxin-3 (mouse monoclonal 1H9, MAB5360, 1:500–1000, Millipore-Sigma); anti-tubulin (mouse monoclonal T5168, 1:10,000, Millipore-Sigma); anti-lamin (mouse monoclonal ADL84.12-5, 1:1000, Developmental Studies Hybridoma Bank); anti-FLAG (rabbit monoclonal D6W5B, 1:500, Cell Signaling Technology); anti-ubiquitin (HRP-conjugated, mouse monoclonal A-5, sc-166553, 1:1000, Santa Cruz Biotechnology); peroxidase-conjugated secondary antibodies: Goat anti-mouse and goat anti-rabbit, 1:5000, were from Jackson ImmunoResearch.

N2A cells were transfected as described above with BicD2-KIF tail fusions and MBNL-EGFP proteins at a ratio of 2:3 in 4-well chamber slides (Thermo Fisher, 154526). 24 h after transfection, cells were fixed and processed for immunofluorescence as described above with a rabbit anti-FLAG antibody (1:1000, Cell Signaling Technologies, D6W5B) to visualize BicD2-KIF fusions. A collection of tile scans was taken from each condition on a Zeiss LSM 880 Axio Observer microscope with widefield illumination using a Plan-Apochromat 1.3 NA ×40 oil objective. After opening each tile scan image on ImageJ, cells and centrosomes were manually chosen, traced, and cataloged with the ROI Manager using MBNL-EGFP and centrosomal FLAG signal as a mask, respectively. Care was taken to analyze cells that were not overexpressing tail proteins and did not have abnormally asymmetric centrosomes. Centrosome recruitment was quantified by taking the log₂ value of the average intensity of MBNL signal at the centrosome divided by the average MBNL intensity in the whole cell.

Chromatin immunoprecipitation (ChIP) was performed with a 3×FLAG-tagged OsHOX3 expressed in callus. About 2 g callus from the 3×FLAG-OsHOX3-overexpressing line was collected and cross-linked with 1% formaldehyde for 15 min. Then the samples were sonicated for isolation and lysis of nuclei. Antibodies against FLAG (D6W5B, Cell Signaling Technology, 1:50 dilution) were used for immunoprecipitation, and Protein-A + G-agarose beads were used to pull down the protein–DNA complex. After collection, wash, and elution of the immune complex, the precipitated DNA was reversely cross-linked and used for qPCR analysis^{65 (link)}. Primers were designed in the promoter regions of OsPYL5 and listed in Supplementary Data 8. Three biological replicates were measured independently.