Rho A Antibody (26C4): sc-418

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

MDCK cells were grown for 5 days and then lysed and subjected to pull-down assays to measure the active GTP-loaded RhoA GTPase, as previously described80,81. Briefly, cell lysates were incubated at 4 °C in assay buffer with 10 µg of GST-rhotekin-RBD immobilized on glutathione-sepharose beads. Active RhoA was pulled-down and total RhoA and GST pull-down samples were subjected to SDS-PAGE and transferred onto immobilon membranes (BioRad). After blocking with 5% BSA (wt/vol) and 0.05% (vol/vol) Tween-20 in Tris-buffered saline, membranes were incubated overnight with the indicated antibodies, washed with Tris-buffered saline containing 0.05% Tween 20, and incubated for 30 min with the corresponding secondary antibodies coupled to HRP. The signal was visualized with Clarity Western ECL substrate (BioRad). Band intensities were quantified using Fiji and the results were represented relative to controls.

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