After boiling in SDS-PAGE loading buffer, the samples were separated on a 12% SDS-PAGE and transferred to nitrocellulose membranes (Whatman GmbH, Germany). Membranes were blocked in 3% non-fat milk-PBS with 0.2% Tween 20 for 1 hr at room temperature or overnight at 4°C. unpurified hs2dAb were used at 1/100 from culture supernatant and added to the membranes with an anti-hisTag antibody at 1/3000 (Sigma-Aldrich) for 90 min. Blots were then washed and incubated 1 hr with secondary anti-Mouse HRP labeled antibodies (diluted at 1/10000 in PBS 0.1% Tween 20) (Jakson ImmunoResearch Laboratories). After 5 washes with PBS 0.1% Tween 20, secondary antibodies were then revealed using the SuperSignal chemoluminescent reagent (Pierce) and Hyperfilm ECL (GE HealthCare). For RHO-GTP pull down, the primary anti RHOA mAb was used (Cell Signaling Technology; 1/1000). For protein knockdown experiments, 500 000 of transfected cells (mCherry positive cells) were sorted with a MoFlo Astrios flow cytometer (Beckman Coulter). Cells were lysed with SDS-Tris lysis buffer (Tris pH7.4 10 mM, SDS 1% supplemented with phosphatase and protease inhibitors). 20 µg of cell extracts were separated on 12.5% SDS-PAGE and electro transferred onto PVDF membranes. Blots were probed with a rabbit polyclonal anti-EGFP full length (Santa Cruz, sc-8334, 1:500), a mouse monoclonal anti-α-tubulin (Sigma, T5168, 1:25000) and an anti-myc HRP antibody (Novus Biologicals, NB600-341, 1:40000). Detection was performed using peroxydase conjugated secondary antibodies and Pierce ECL Western Blotting Substrate (Thermo Scientific Pierce).

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