Rhoa rac1 cdc42 activation assay combo kit

The pull-down assays were performed using the RHOA/RAC1/CDC42 Activation Assay Combo Kit (Cell BioLabs, San Diego, CA, USA) following the manufacturer’s instructions. Briefly, cell lysates were incubated with GST-RBD (for RHOA pull-down) or GST-CRIB (for RAC1/CDC42 pull-down) for 1 h at 4 °C with gentle agitation. Then, the samples were centrifuged, and the pellet was washed several times. After the last wash, the pellets were resuspended with sample buffer and boiled for 5 min. GTP-RHOA, GTP-RAC1, and GTP-CDC42 were detected by Western blotting using anti-RHOA, anti-RAC1, and anti-CDC42, respectively. Total RHOA/RAC1 and CDC42 were collected prior to the incubation with GST-RBD/GST-CRIB and used as a loading control. All Western blots were analyzed with ECL (GE Healthcare, Chicago, Il, USA) followed by densitometry.

Levels of GTP-bound Rac1, GTP-bound CDC42 and GTP-bound RhoA were detected using an RhoA/Rac1/Cdc42 Activation Assay Combo Kit (Cell Biolabs, #STA-405) according to the manufacturer’s recommendations. In briefly, indicated cells were starved in serum-free medium for 24 h and stimulated with 10% FBS for 2 h, then collected by lysis buffer. Cell lysates were incubated with Rhotekin RBD or PAK1 PBD agarose beads for 1 h at 4 ℃. Immunoblotting was used to detect GTP-RhoA, GTP-RAC1 and GTP-CDC42.

S1 and Cx43-shRNA S1 cells were plated in T-75 tissue culture flasks (2-D) or 35-mm tissue culture plates (3-D). Acini were isolated from 3-D cultures as previously described [60 (link)]. The pulldown assays were performed using the RhoA/Rac1/Cdc42 Activation Assay Combo Kit (Cell Biolabs, San Diego, CA, USA, STA-405) following the manufacturer’s instructions. Briefly, lysates of cells on day 9 (2-D) and acini on day 11 (3-D) were incubated with PAK PBD agarose beads (for Rac-GTP and Cdc42-GTP pulldown) for 1 h at 4 °C with gentle agitation. The samples were then centrifuged, and the pellets were washed several times. Subsequently, the pellets were resuspended in Laemmli sample buffer and boiled for 5 min. GTP-Rac1 and GTP-Cdc42 were detected by Western blotting using mouse monoclonal antibodies against Rac1 or Cdc42, respectively. Lysates were collected prior to the incubation with PAK PBD agarose beads and were immunoblotted with mouse monoclonal antibodies against RhoA, Rac1, and Cdc42 for detection of total GTPase levels.

Small GTPase pull-down assay was performed using the RhoA/Rac1/Cdc42 Activation Assay Combo Kit (Cell BioLabs, San Diego, CA, USA) following the manufacturer's instructions. Cells were incubated with medium and 1 ng/mL EGF for 2–4 days until they obtained approximately 80–90% confluence. Cell lysates were incubated with Rhotekin RBD (for RhoA) or PAK1 PBD (for Rac1/Cdc42) agarose beads for 1 hour at 4°C. GTP-RhoA, GTP-Rac1, or GTP-Cdc42 was detected by immunoblotting.

Cells were either transfected with GFP-StarD13 construct or an empty GFP construct as a control. Following treatment period, cells were lysed and the pull-down assay performed using the RhoA/Rac1/Cdc42 Activation Assay Combo Kit (Cell BioLabs) following the manufacturer’s instructions. Briefly, cell lysates were incubated with GST-RBD (for RhoA) or GST-PAK (for Rac1/Cdc42) for 1 h at 4˚C with gentle agitation. Then, the samples were centrifuged, and the pellet washed for several times. After the last wash, the pellets were resuspended with sample buffer and boiled for 5 min. GTP-RhoA and GTP-Rac1/Cdc42 were detected by western blotting using anti-RhoA, anti-Rac1 and anti-Cdc42 antibodies provided in the kit. Total proteins were collected prior to the incubation with GST beads and used as a loading control.