HEK293T cells were fixed and stained as previously described (Coyne et al., 2020 (link)) using Rabbit anti-phospho-UPF1 antibody (Millipore Sigma) at a 1:250 dilution and AlexaFluor 488 conjugated Goat anti-Rabbit secondary antibody (Thermo Fisher Scientific) at a 1:1000 dilution in a 24-well glass-bottom plate (Cellvis). 9 sites in each well were imaged using a 20x objective on an ImageExpress Micro Confocal High-Content Imaging System (Molecular Devices).
iPSNs were fixed with 4% (v/v) para-formaldehyde in PBS for 10 min, permeabilized in 0.2% (v/v) Triton X-100 for 10 min, blocked in 1% bovine serum albumin and 2% goat serum for 1h, incubated with primary antibodies overnight at 4°C, washed with PBS, and finally incubated with Alexa Fluor 488/647 conjugated secondary antibodies (ThermoFisher Scientific). Cells were imaged with a Zeiss 800 Airyscan microscope.
iPSNs were fixed with 4% (v/v) para-formaldehyde in PBS for 10 min, permeabilized in 0.2% (v/v) Triton X-100 for 10 min, blocked in 1% bovine serum albumin and 2% goat serum for 1h, incubated with primary antibodies overnight at 4°C, washed with PBS, and finally incubated with Alexa Fluor 488/647 conjugated secondary antibodies (ThermoFisher Scientific). Cells were imaged with a Zeiss 800 Airyscan microscope.