Duolink in situ detection reagent kit

MCF7 cells transduced with the GEF dox-inducible system were seeded on glass coverslips in the presence of 1 µg ml ⁻¹ dox for 24 hours. Cells were then fixed in 4 % formaldehyde and subjected to the Duolink in situ Proximity Ligation Assay (PLA). Mouse anti-Rac1 (BD Biosciences, 610650) and rabbit anti-TMOD3 (Sigma-Aldrich, HPA001849) were used together with the respective Duolink in situ PLA probes (Olink Bioscience, anti-mouse 92004-0100, anti-rabbit 92002-0100) and the Duolink in situ detection reagent kit (Olink Bioscience, 92013-0100, 92014-0100) according to manufacturer's instructions. Coverslips were mounted onto slides using ProLong Gold antifade reagent with DAPI stain (Life Technologies, P36935). Phalloidin and DAPI were used to visualize the actin cytoskeleton and the nuclei, respectively. Images were captured on the Low Light microscope system using fixed focus and exposure settings to ensure that differences detected in the Duolink signal are only due to changes in Rac1-TMOD3 binding. Quantification of the Duolink signal was performed as described by Marei et al.¹⁸ (link)

Cells seeded on glass coverslips in the presence of 1 μg ml⁻¹ dox for 24 h were fixed in 4% formaldehyde and subjected to Duolink in situ PLA using mouse anti-Rac1 antibody (1:100; BD Biosciences, 610650) and rabbit anti-FLII antibody (1:100; Sigma-Aldrich, HPA007084), the respective Duolink in situ PLA probes (Olink Bioscience, anti-mouse 92004-0100, anti-rabbit 92002-0100) and the Duolink in situ detection reagent kit (Olink Bioscience, 92013-0100, 92014-0100) according to manufacturer's instructions. For CHL1 cells, mouse anti-FLII antibody (1:100; Santa Cruz, sc-21716) and rabbit anti-P-Rex1 antibody (1:100; Sigma-Aldrich, HPA001927) were used for the Duolink in situ PLA analysis. Coverslips were mounted onto slides using ProLong Gold antifade reagent with DAPI stain (Life Technologies, P36935) and images were taken using the low light microscope system (× 100 magnification). Phalloidin and DAPI were used as fluorescence markers against the actin cytoskeleton and the nucleus, respectively. Quantification of the Duolink signal was performed as outlined in Supplementary Methods.