Rac1 activation assay kit

5d, which purity > 99%, was synthesized by the Center of Drug Discovery, China Pharmaceutical University. Its molecular structure was shown in previous studies [9 (link), 29 (link)] and was dissolved in normal saline. Membrane and cytosol protein extraction kit and Lactate dehydrogenase (LDH) kit were obtained from Beyotime Institute of Biotechnology (Haimen, China). Rac1 activation assay kit was purchased from Millipore Corporation (MA, USA). The primary antibodies used were polyclonal antibodies against CK2α, CK2α', CK2β, NOX4, Rac1, p47^phox, p67^phox, β-actin (Millipore Corporation, MA, USA). The goat anti-rabbit IgG-HRP secondary antibody was supplied by Bioworld Technology Inc. (MN, USA). NBP, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and all other chemicals were purchased from Sigma Chemical Co., (St. Louis, MO, USA).

BM cells from corresponding mice at 7 DPI of pIpC were collected and immediately subjected to Rac1 activation assay with the Rac1 activation assay kit (Millipore) according to the manufacturer's instructions.

Assays were performed using a Rac1 Activation Assay kit, according to the manufacturer's recommendations (Millipore). Cells were cultured to approximately 70% confluence and then serum-starved for 24 hr. Rac1 activation was provoked by adding complete culture medium for 24 hr. The cells were lysed in Mg²⁺ lysis buffer containing protease inhibitors and phosphatase inhibitors, followed by centrifugation to remove cell debris. Equal amounts (0.5mg) of total protein were incubated with 10 μl of agarose-conjugated p21-binding domain of PAK1, which binds activated Rac1, for 1 hr at 4°C. The agarose beads were washed three times in lysis buffer, resuspended in 20μl 4× SDS loading buffer, and boiled for 10 min. Active (GTP-bound) and total Rac1 were analyzed by Western blotting.

Western blots were performed as previously described [17] . Briefly, neurospheres (after 6 days in culture) were lysed in MAPK lysis buffer (20 mM Tris (pH 7.0), 10 mM EGTA, 400 mM B-glycerophosphate, 1% NP-40, 2.5 mM MgCl₂, and 2 mM sodium orthovanadate with protease inhibitors (leupeptin, aprotinin, and phenylmethylsulfonyl fluoride). For all experiments, 30 µg of protein per sample were loaded in each lane. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA) prior to detection with phospho-specific antibodies. Antibodies that detect the total expression of the corresponding signaling intermediate as well as α-tubulin were used as controls for equal protein loading and quantitation. Active Rac1 (Rac1-GTP) was determined by PAK1-PBD affinity chromatography (Rac1 activation Assay Kit, Millipore, Bedford MA) according to the manufacturer’s recommendations. Appropriate HRP-conjugated secondary antibodies (Cell Signaling, Beverly MA) were used for detection by enhanced chemiluminescence (New England Biolabs, Beverly MA). Antibodies used are listed in Table S2.

Rac1 activity was assessed using the Rac1 Activation Assay Kit (Millipore) according to the manufacturer’s instructions. Briefly, cell lysates were clarified by centrifugation at 14,000 ×g at 4°C for 10 min. Equal volumes of lysates were incubated with beads to pull down activated Rac1 proteins. After incubation at 4°C for 1 h, the beads were washed three times with cold MLB buffer. The Rac1 proteins were eluted with sample buffer and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Western blot was analyzed using anti-Rac1 antibodies (Millipore). GAPDH was used as a housekeeping gene for normalization.