Anti rac1 antibody

GST and GST-SH3 proteins were expressed in Escherichia coli BL21 and purified using Glutathione Sepharose 4B (#17-0756-01; GE Healthcare, Buffalo Grove, IL, USA), as described previously [27 (link), 28 (link)]. HEK293T cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed with pull-down buffer (50 mM Tris-Cl, pH 7.4, 100 mM NaCl, 0.5 % Triton X-100, 5 % glycerol, 5 mM MgCl₂, 1 mM DTT, 20 mM NaF, 1 mM aprotinin, 1 mM leupeptin, and 1 mM pepstatin). After centrifugation at 21,000 × g for 15 min at 4 °C, the supernatant was incubated with 5 µg GST or GST-SH3 protein in pull-down buffer for 1 h at 4 °C and combined with 20 µl Glutathione Sepharose 4B. After incubation, the beads were washed three times with pull-down buffer, after which Western blotting was performed. For the GST-PBD pull-down assay, GST-PBD purified protein, a p21-binding domain of PAK1, was employed. HEK293T cells transfected with the indicated vectors were lysed with pull-down buffer and incubated with 5 µg GST-PBD protein for 1 h at 4 °C, followed by incubation with 20 µl Glutathione Sepharose 4B. Active Rac1 was pulled down and visualized using Western blotting with an anti-Rac1 antibody (#610651; BD Transduction Laboratory).

PAK-PBD beads were purchased from Cytoskeleton Inc. Cells were lysed using Rac1 IP buffer (50 mM Tris-HCl [pH 7.5], 10 mM MgCl₂, 0.5 M NaCl, and 2% Nonidet P-40), and the protein concentration was adjusted to 0.5 mg/ml. For the pull-down assay, 800 μl of cell lysate was used for each time point, and 10 μl of PAK-PBD beads was added to each lysate. Bound GTP-Rac1 was detected by Western blotting using an anti-Rac1 antibody (BD Biosciences).

Protein extraction and western blotting were carried out using standard protocols^{54 (link),55 (link)}. β-Actin detection using a rabbit mAb (Cat# 8457 (D7A8); Cell Signaling), GRB2 (Cat# 610111; BD Transduction Laboratories), GAPDH (Cat# 2118; Cell Signaling), or Akt (Cat# 4691; Cell Signaling) were used as loading control. Hemagglutinin (HA) epitope tag monoclonal antibody (anti-HA antibody, Cat# MMS-101P-1000 (1.0 ml), Covance (Cedarlane)) recognizing an HA-epitope tagged HACE1 protein and anti-HACE1 antibody (Cat# ab133637; Abcam), that detect different HACE1 fragments, were used at 1:1000 dilution. Western blotting for total RAC1 was conducted using an anti-RAC1 antibody (Cat# 610650; BD Biosciences), and RAC1/2/3 was assessed using an anti-pan-Rac antibody (Cat# 2465; Cell Signaling). All antibodies were used at a dilution of 1:1000 unless otherwise stated.