Rac1 activation assay biochem kit

Relative levels of GTP-bound Rac1 were determined by Rac1 Activation Assay Biochem Kit™ (Cytoskeleton) according to manufacturer’s procedure. Briefly, BLA were isolated and homogenized in IP lysis buffer (Beyotime Biotechnology) containing phosphotransferase inhibitor. Lysates were precipitated with PAK-PBD affinity beads for 4 h at 4°C, and then bound Rac1 protein was eluted from pelleted beads. Activated Rac1-mCherry and total Rac1-mCherry were examined by Western Blot with mCherry antibody (1:1000, Rockland), and endogenous activated and total Rac1 were detected with a Rac1 specific antibody (1:2000, BD Transduction Laboratories). For p-Cofilin and p-ERK measurement, cultured astrocytes expressing PA-Rac1 were irradiated for 5 min at 473 nm wavelength (6 mV), then lysed immediately with cold lysis buffer for Western Blot. All procedures were done at dark room to avoid unspecific light-activation of Rac1. For detecting Cofilin and ERK, antibody were used as follows: Cofilin (1:1000, CST), p-Cofilin (1:500, CST), ERK (1:2000, CST), p-ERK (1:2000, CST). Intensities of the detected bands in Western Blots were quantified in ImageJ software.

Cells lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and the proteins were transferred to a nitrocellulose membrane. The membrane was hybridized with antibodies against pRb, phosphorylated pRb at Ser 795, Akt, phosphorylated Akt at Ser 473, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, PARP (Cell Signaling, Danvers, MA, USA), Mcl-1, unprenylated Rap1A, Rab6 (Santa Cruz Biotech, Santa Cruz, CA, USA), RhoA, Cdc42, Rac1 (Cytoskeleton), Ras (BD Biosciences) or actin (Sigma-Aldrich). The membranes were developed with the ECL system (GE Healthcare, Buckinghamshire, UK). Membrane and cytoplasm fractions were separated with a native membrane extraction kit (Merck Millipore) according to the manufacturer's protocol. Intensity of hybridized bands was determined with the public domain Image J program (available at http://rsbweb.nih.gov/ij). GTP-binding RhoA and Rac1 proteins were isolated with a pull-down assay using a Rho activation assay biochem kit and a Rac1 activation assay biochem kit (Cytoskeleton) according to the manufacturer's protocol.

The RAC1 pull‐down assay was constructed according to the manufacturer's instructions for the Rac1 Activation Assay Biochem Kit (BK035, Cytoskeleton, Denver, USA). Briefly, cell lysates were collected for Rac1 activation studies when the cells had grown to approximately 70% confluence. Then, 400 µg protein lysate was added to a predetermined 10 µg amount of PAK‐PBD beads. The mixture was incubated at 4°C on a rotator for 1 h. The supernatant was carefully removed after the PAK‐PBD beads were pelleted by centrifugation, and the beads were washed once with 500 µL of wash buffer. Then, 10‐20 µL of 2× Laemmli sample buffer was added to each tube, and the bead samples were boiled for 2 min. Finally, the samples were analyzed by SDS‐PAGE and Western blot analyses with an anti‐Rac1 antibody.

For Rac1-activation analysis, the cells were washed with PBS and subsequently lysed in cell lysis buffer, provided with the “Rac1 Activation Assay Biochem Kit” (Cytoskeleton, Denver, USA). The protein concentration was determined using the “BCA Protein Assay Kit” (Pierce, Thermo Fisher Scientific) and 350 µg of the cell lysates was incubated at 4 °C with 10 µl of the “PAK-PBD beads” on a rotator for 1 h. The “PAK-PBD beads” were pelleted (1 min, 4 °C, 5000 × g), the supernatants carefully removed, and analyzed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and total Rac1 in Western blot. The beads, carrying the specifically bound Rac1-GTP, were resuspended with 500 µl wash buffer. The beads were again pelleted (3 min, 4 °C, 5000 × g), the pellets resuspended in water, sample buffer added, and the samples analyzed in Western blot. The measurement of RhoA was performed accordingly, also as suggested by the manufacturer (Cytoskeleton).

Rac1 and RhoA activity were assessed using Rac1 Activation Assay Biochem Kit (Cytoskeleton, #BK035) and Rho Activation Assay Biochem Kit (Cytoskeleton, #BK036), respectively, following the manufacturer’s instructions. In brief, Rac1-GTP and RhoA-GTP were pulled down from total proteins using the matching beads (PAK-PBD beads for Rac1 and Rhotekin RBD beads for RhoA), then the total and active Rho GTPase was detected by Western blot with anti-Rac1 antibody (Cytoskeleton) and anti-RhoA antibody (Cytoskeleton).