Tyr1173

After drug treatment, cells were washed with cold PBS and lysed using SDS lysis buffer (100 mM Tris, 1% SDS, 10% glycerol). Lysates were boiled at 100 °C for 5 min. Protein concentrations were measured using BCA Protein Assay Reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. Equal amounts of protein were separated by SDS–PAGE and transferred to polyvinylidene difluoride membranes. Antibodies against phospho-EGFR (Tyr 1068; GTX132810, GeneTex, 1:2000, or Tyr1173; Cell Signaling Technology, #4407, 1:1000), total EGFR (Cell Signaling Technology, #4267, 1:2000), phospho-Akt (Ser473; Cell Signaling Technology, #4060, 1:1000), total Akt (Cell Signaling Technology, #4691, 1:5000), phospho-ERK (Thr202/Tyr204; Cell Signaling Technology, #9101, 1:5000), total ERK1/2 (Cell Signaling Technology, #9102, 1:2000), phospho-S6 (Ser240/244; Cell Signaling Technology, #5364, 1:10,000), total S6 (Cell Signaling Technology, #2217, 1:2000), and β-actin (Sigma-Aldrich, A5228, 1:10,000 or Sigma-Aldrich, clone; AC-15, 1:10,000) were used. Uncropped Images are presented in Supplementary Fig. 10.

Paraffin sections were stained with mouse anti-FUS (sc-47711, Santa Cruz Biotechnology), rabbit anti-GFP (NB600-308, Novus Biological), rabbit anti–phospho-EGFR (Tyr1173; 4407, Cell Signaling Technology), or rabbit anti–collagen IV (600401106, Rockland) antibodies followed by Alexa Fluor 488 anti-mouse (A32723, Alexa Fluor) and Alexa Fluor 555 anti-rabbit (A21428, Alexa Fluor). Slides were then mounted using ProLong Gold Antifade Mountant with DAPI (P36931, Thermo Fisher Scientific).
Images were taken using a Leica DM6000B upright microscope (×20 or ×40 lens) equipped with a Leica EC4 microscope camera, and images were captured using LAS X Widefield Systems software.
For kidneys, the number of FUS-positive cells per glomerulus and the intensity of collagen IV per glomerulus were analyzed using ImageJ (NIH), and values were expressed as percentage of FUS-positive cells among total glomerular cells or collagen IV intensity per glomerulus.
For livers, FUS nuclear intensity was analyzed using CellProfiler, and values were expressed as FUS nuclear intensity per microscopic field. Collagen IV intensity was analyzed as described above and expressed as collagen IV intensity per microscopy field.