G lisa rac1 activation assay biochem kit

Sperm RAC1-GTP levels (a proxy for RAC1 activity) were compared between +/+ (n = 6), t/+ (n = 4) and t/t (n = 6) mice by using the luminescence-based G-LISA Rac1 activation assay biochem kit (Cytoskeleton, Inc., Denver, CO, USA), according to the manufacturer´s guidelines. Samples were washed in Dulbecco’s Phosphate Buffered Saline (DPBS; Lonza, Basel, Switzerland) and sperm pellets were used to prepare lysates. A preliminary experiment was performed in order to check that the assay was in the linear range. Extracts were equalized to 0.4 mg/ml total protein. Samples were run in triplicates and readings were done using a GloMax multi-detection system (Promega, Madison, WI, USA). Only triplicate values with low variance were accepted.

Dehydroisoandrosterone 3′-sulphate (cat no. D5297), hematoxylin (cat no. H3136), anti-beta-actin (cat no. A2668) and goat anti-Mouse IgG (γ-chain specific)-HRP (cat no. A3673) were purchased from Sigma Aldrich Inc., St Louis, MO, USA. Immobilon-P PVDF membrane (0.45 μm), ECL reagent kits (cat no. WBKLS0500), Protein-A-Agarose suspension (cat no. IP02) and goat anti-rabbit-HRP IgG (cat no.621140380011730) were procured from Merck-Millipore, Cedex, France. Other primary antibodies against phospho-Vav (Y174) (cat no. ab47282), phospho-Rac1/Cdc42 (S71) (cat no. ab5482) and Rac1 (cat no. ab33186) were purchased from Abcam, Cambridge, MA, USA. Anti-Vav (cat no. sc132) and anti-Caveolin1 (cat no. sc894) were purchased from Santa Cruz Biotechnology, CA, USA. Non-fat milk (cat no.170-6404) and precision plus protein standard marker (cat no. 161-0374) were obtained from Bio-Rad Lab., Inc., Hercules, CA., USA. Protein assay kit (cat no. 23225) was procured from Thermo-Scientific, Rockford, USA. The G-LISA Rac1 activation assay Biochem Kit (cat no. BK128) was purchased from Cytoskeleton, Denver, CO, USA. Inhibin B Enzyme Immunoassay kit (cat no. EIA-INB-1) was purchased from RayBiotech, Inc., Norcross, GA, USA. 17 β-estradiol assay kit (cat no. ADI-900-008) was obtained from Enzo Life Science, Inc., Farmingdale New York, USA.

To analyse the activation of Rac1 in D₂R and WT RPE, G-LISA Rac1 activation assay Biochem Kit (Cytoskeleton, Inc., Denver, CO, USA) was used. Rac1 activation assay was performed following the manufacturer's instructions on fresh lysates of RPE tissue collected at ZT 23, ZT 1, and ZT 3, as mentioned previously (Baba et al., 2010).

KDR cells at 50%confluence were incubated for 24 h in FCS-free DMEM to starve the cells, and then treated with 10% FCS for 30 min. Cell lysates were collected and levels of activated GTP-bound Rac1, RhoA and Cdc42 were determined using the G-LISA Rac1 Activation Assay Biochem Kit (BK126, Cytoskeleton Inc., CO, USA), G-LISA RhoA Activation Assay Biochem Kit (BK121, Cytoskeleton Inc.) and G-LISA Cdc42 Activation Assay Biochem Kit (BK127, Cytoskeleton Inc.), respectively.

The antibodies we used were anti-E2F1 (KH95; Santa Cruz Biotechnologies), anti–epidermal growth factor (anti-EGFR) (1005; Santa Cruz Biotechnologies), anti-ERα (SP-1; Abcam), antibody against phosphorylated extracellular signal-regulated protein kinases 1 and 2 (anti-phospho-ERK1/2) (D13.14.4E; Cell Signaling Technology, Danvers, MA, USA), anti-NUP62 (nucleoporin 62 kDa, clone 53; BD Transduction Laboratories, San Jose, CA, USA), anti-PAK1 (2602; Cell Signaling Technology), anti-RAC1 (05-389; EMD Millipore), anti-phospho-serine 235/236 ribosomal S6 (D57.2.2E; Cell Signaling Technology), anti-VAV3 (07-464, Millipore; and 2398, Cell Signaling Technology), anti-phospho-tyrosine 173 VAV3 (anti-pT173 VAV3, ab52938; Abcam) and anti–tubulin α (anti-TUBA) (DM1A + DM1B; Abcam). Secondary antibodies for used for immunofluorescence (Alexa Fluor) were obtained from Molecular Probes (Eugene, OR, USA). To measure RAC1 activity, we used the Rac1 G-LISA Activation Assay Biochem Kit (BK128; Cytoskeleton, Denver, CO, USA). The MYC-Vav3 wild-type and oncogenic expression constructs we used have been described previously
[30 (link),31 (link)].