Active rho pull down and detection kit

The level of active Rho in wild-type or Dlc1-KO MEFs was measured using the Active Rho Pull-down and Detection kit (Thermo Scientific). Briefly, a GST-fusion of the Rhotekin binding domain (RBD) was used to pull down active Rho (Rho-GTP) and the eluted sample was probed using Western Blot with an anti-RhoA antibody (1:200, Santa Cruz) to identify the amount of active RhoA.

Endogenous active guanosine triphosphate (GTP)-bound RAC1 and RhoA were pulled down using Active Rac1 Pull-Down and Detection Kit (16118, Thermo Scientific™, Pierce, USA) and Active Rho Pull-Down and Detection Kit (16116, Thermo Scientific™, Pierce, USA) according to the manufacturer’s protocol [33 (link)].

The assays were performed with the Active Rho Pull-Down and Detection kit (CAT#: 0016116, Thermo Fisher Scientific) according to the manufacture's instruction. In brief, cells were lysed by 1×lysis buffer and the same amount of total proteins was incubated with agarose beads and GST-Rhotekin-RBD protein-binding domain. After incubation at 4°C for 1 h, the samples were washed three times with 0.4 ml of 1× lysis buffer. Following the last wash, 50 μl 2× reducing buffer was added to the samples and incubated at room temperature for 2 min. At last the samples were collected by centrifugation at 6000g for 2 min and were electrophoresed on an SDS-PAGE gel followed by western blot analysis.

Active Rho were measured using the pull-down method with Active Rho Pull-Down and Detection Kit (Thermo Fisher, Waltham, MA, USA), according to the manufacturer’s instructions. Briefly, GTP-bound Rho were pulled down by the incubation of cell lysates with GST-Rhotekin-RBD protein agarose beads, then detected by Western blot analysis.

HT-29 cells were washed with PBS, and then lysed with 1% NP-40 on the culture dishes. Active Rho Pull-Down and Detection Kit (Thermo Scientific, 16116) was used to measure the level of active RhoA, according to the manufacturer’s instructions. GST-Rhotekin-RBD bound proteins were separated by SDS-PAGE and then immunoblotted with an anti-RhoA antibody.