Rac1 cdc42 activation assay kit

G418, Fetal bovine serum (FBS), phallotoxins, RPMI 1640, DMEM, non-essential amino acids (NEAA), subcellular protein fractionation kit for cultured cells and Lipofectamine^® 2000 Transfection Reagent were from Thermo Fisher (Waltham, MA, United States). Transwell^® polycarbonate membrane cell culture inserts were bought from Corning (Tewksbury, MA, United States). Anti-vimentin (ab8978), -CCNY (ab107012), -snail (ab167615), -PFTK1 (ab224098), -β-catenin (ab6302), -TPM4 (ab181156), and -ZEB1 (ab124512) antibodies were purchased from Abcam (Cambridge, United Kingdom). The Rac1/Cdc42 activation assay kit and RhoA activation assay kit were obtained from Millipore (Billerica, MA, United States). Hoechst 33342 were obtained from Sigma (St. Louis, MO, United States). Matrigel Matrix was purchased from BD Science (San Diego, CA, United States).

Control (scrambled) or MafB-KD MS1 ECs were serum starved for 2 h and stimulated with complete EGM-2 media for 30 min. Active Rac1 and Cdc42 were analyzed with Rac1/Cdc42 Activation Assay Kit (Millipore) according to the manufacturer’s instructions. An aliquot of 100 μM of GTPγS or 1 mM of GDP were added to cell extracts and incubated at 30 °C for 15 min with agitation for positive or negative control, respectively.

Rac1/Cdc42 activity was assessed using the Rac1/Cdc42 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 analysis was performed using anti-Rac1 or Cdc42 antibodies (Millipore).

The rabbit polyclonal anti-mPRβ antibody (bs-11410R; Bioss Inc) was used to immune-precipitate mPRβ. TrkA was immunoprecipitated using the rabbit polyclonal anti-TrkA antibody (#2505; CST) as described previously^{2 (link)}. To detect Rac-1 (Rac-1-GTP) in cell lysates, we used a Rac-1/Cdc-42 Activation Assay Kit (17-441, Millipore), using the manufacturer’s instructions. Cells were washed three times with ice-cold PBS and collected by gently scraping using 1 mL of ice-cold MLB Buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl₂, 1 mM EDTA and 10% glycerol, aprotinin 10 μg/ml).

To measure the activity of RhoA, Rac1, and Cdc42, 96-well plates were coated with methanol-solubilized nitrocellulose and air-dried under a sterile hood. PLL coating on the nitrocellulose-coated surfaces of the wells was performed to promote microglia adhesion. Then, BSA (0.01% in PBS), fAβ_1–42 (10 μM), Nogo-P4 (100 μg/ml), or Nogo-P4 (100 μg/ml) + fAβ_1–42 (10 μM) were coated on the wells overnight at 4 °C. Adult microglia were added into the pre-coated wells and cultured for 6 h.
The activity of RhoA, Rac1, and Cdc42 were determined by commercial Rho Activation Assay Kit (Millipore) and Rac1/Cdc42 Activation Assay Kit (Millipore) following the manufacturer’s instructions.

Rabbit polyclonal anti-MEC-17 (#ab58742), anti-GM130 (#ab52649), and anti-GFP (#ab6556) antibodies were purchased from Abcam (Cambridge, MA). Mouse monoclonal anti-acetylated tubulin (#T7451) and anti-α-tubulin (#T6199) antibodies were obtained from Sigma-Aldrich (St. Louis, MO). Monoclonal anti-actin (#MAB1501), anti-cdc42 (#07–1466), and a Rac1/cdc42 activation assay kit (#17–441) were purchased from Millipore (Temecula, CA). Anti-E-cadherin (#610182) and anti-N-cadherin (#610920) antibodies were purchased from BD Transduction Laboratories. Anti-GAPDH (#2118) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Antibody against vimentin (#sc-32322), anti-ARHGAP21 (#sc-390145), anti-mouse IgG (#sc-2060), and anti-rabbit IgG (#sc-2054) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa-Fluor-488-conjugated anti-rabbit IgG (#A11008) and Alexa-Fluor-568-conjugated anti-mouse IgG (#A11004) were purchased from Molecular Probes (Eugene, OR). T4 DNA ligation kit and restriction enzymes were purchased from New England Biolabs (UK) or Thermo Scientific (Rockford, IL). RIPA buffer solution was purchased from Thermo Scientific. Cell culture reagents were obtained from Life Technologies, Inc. (Rockville, MD).

Rac1 activation assay was previously described (54 (link)). In brief, 2 × 10⁵ cells were seeded on collagen-coated six-well plates (Cellmatrix Type 1-C), cultured for the indicated time in either the presence or absence of 1 ng/ml TGF-β1, and harvested. Rac1 activation was determined by a GST pulldown assay using a Rac1/Cdc42 activation assay kit (Millipore 17-441).