How the TREX-2 complex associates with the nuclear pore, SMPD4 immunofluorescence
Description
Fig. S4a. As part of the manuscript: How the TREX-2 complex associates with the nuclear pore (Obarska-Kosinska et al.) Immunofluorescence images of HEK293 cells stained with rabbit polyclonal antibody against human SMPD4 (Sigma-Aldrich, HPA049426) and secondary goat anti-rabbit IgG antibody conjugated to Alexa Fluor 680 (Thermo Fischer Scientific, A-21109).
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For immunofluorescence staining, adherent human embryonic kidney (HEK293) cells were cultured on laminin-coated ibidi 18-well dishes (Roche, 11243217001). Cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature (RT), permeabilized with 0.1% Triton X-100 in PBS for 5 min at RT, and blocked for 1 h at RT in blocking buffer (2% BSA and 0.1% Tween-20 in PBS). Cells were incubated with a rabbit polyclonal antibody against human SMPD4 (Sigma-Aldrich, HPA049426) diluted 1:75 in 1 % BSA in PBS for 1 h at RT, followed by incubation with a goat anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 680 (Thermo Fischer Scientific, A-21109) diluted 1:1000 in 1 % BSA in PBS for 1 h at RT. Nuclei were stained with DAPI (0.25 µg/mL in PBS) for 5 min at RT. The samples were sealed with Prolonged Antifade Glass mounting medium (Invitrogen). Images were acquired on an inverted Stellaris 5 confocal microscope (Leica) using a 63×/1.4 NA oil-immersion objective and processed in FIJI.