α tiar

U-2 OS cells were grown in 8-well chamber slides (Ibidi, Fitchburg, WI, USA, 80821), fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Trion X-100 for 10 min and then blocked in 5% goat serum for 1 h. Cells were incubated with indicated primary antibodies at RT for 2 h. Cells were washed three times and then incubated with Alexa Fluor conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA, 1:1000 dilution) for 1 h at room temperature. After three washes with PBS, cells were stained with DAPI (Thermo Fisher Scientific, Waltham, MA, USA, P36931) and imaged using a Deltavision Elite microscope (Cytiva, Marlborough, MA, USA) with a 20× objective. Antibodies used for immunofluorescence were α-TDP-43 (Proteintech, Rosemont, IL, USA, 60019-2-Ig dilution 1:200, Proteintech, Rosemont, IL, USA, 10782-2-AP dilution 1:200), α-phospho(S409/S410)-TDP43 (Proteintech, Rosemont, IL, USA, 22309-1-AP dilution 1:200), α-G3BP (Abcam, Cambridge, MA, USA, ab181149 dilution 1:500), α-TIAR (Santa Cruz, Dallas, TX, USA, sc-398373 dilution 1:200). G3BP1/2ΔΔ cell lines were generated as previously described [32 (link)].