Visualizing Rac1-TMOD3 Interactions in MCF7 Cells

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)

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Marei H., Carpy A., Macek B, & Malliri A. (2016). Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors. Cell Cycle, 15(15), 1961-1974.

Publication 2016

HPA001849 Duolink in situ PLA probes Duolink in situ detection reagent kit ProLong Gold antifade reagent with DAPI stain

Actin cytoskeleton Assay Cells Dapi Formaldehyde Gold Ligation Mcf7 cells Microscope light Mouse Nuclei Phalloidin Rabbit Stain

Corresponding Organization : University of Manchester

Other organizations : University of Tübingen

Top 5 similar protocols

Variable analysis

independent variables

Dox-inducible GEF system

dependent variables

Rac1-TMOD3 binding (quantified using Duolink PLA signal)

control variables

Cell type (MCF7 cells)
Dox concentration (1 μg/ml)
Dox induction time (24 hours)
Fixation method (4% formaldehyde)
Antibodies used (mouse anti-Rac1, rabbit anti-TMOD3)
Imaging settings (fixed focus and exposure)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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