Additionally, 8-Br-cAMP reversed increases in RhoA activity induced by Sema 3A in whole cell lysates and CSPGs in growth cones. cell body fractions were collected from the AZD7507 sucrose cushion/buffer interface and pellet, respectively. Sucrose was removed by centrifugation (14,000 20 minutes). Each fraction was lysed on ice in 1.0% IGEPAL CA-630, 1.5 mM EDTA, 25 mM Tris-HCl (pH = 7.4), and 150 mM NaCl (Sigma, St. Louis, MO). Each fraction (40 g total protein; BCA, Pierce, Rockford, IL) was electrophoresed through 15% sodium dodecyl sulfate polyacrylamide gels (SDS/PAGE) and transferred to nitrocellulose (BioRad, Temecula, CA). After blocking in 5% non-fat milk (Fisher, AZD7507 Pittsburgh, PA) in Tris buffered saline made up of 0.1% Tween-20 (TBST), membranes were incubated overnight at 4.0C with 2G13 (1:1000), anti-lamin B (1:500, Calbiochem, La Jolla, CA), anti-actin (1:500, Cytoskeleton, Denver, CO) Rabbit polyclonal to MMP1 or anti–tubulin (1:500, Invitrogen, Carlsbad, CA). Membranes were washed with TBST and incubated in goat anti-mouse or goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Invitrogen, Carlsbad, CA) for 2 hours at room temperature. Membranes were washed extensively and immunoreactive bands were visualized by enhanced chemiluminescence (ChemiGlow; Alpha Innotech, San Leandro, CA). Coomassie blue-stained sister gels were used to assess protein loading in both fractions. Rac1 activation in growth cones and whole cell lysates was assessed using pull-down assays. Cells (90% confluent) were maintained in SCM or placed in serum-free medium (SFM) for 24 hours and subsequently treated with outgrowth promoters and inhibitors for 15 minutes. Cells were fractionated and lysed in 1.0% IGEPAL CA-630, 1.5 mM EDTA, 25 mM Tris-HCL (pH = 7.4), and 150 mM NaCl. Growth cone or whole cell lysates (200C400 g total protein) were incubated with 20 g p21-activated kinase protein binding domain name conjugated agarose beads (Cytoskeleton, Denver, CO) for 45 minutes at 4.0 C. Beads were washed three times in 25 mM Tris (pH = 7.5), 30 mM MgCl2, and 40 mM NaCl (all from Sigma, St. Louis, MO) and boiled in Laemmli buffer (Sigma, St. Louis, MO). Samples (20C40 g lysates; entire pull-down) were immunoblotted with mouse (1:500; BD Biosciences, San Jose, CA) or rabbit (1:500; Cell Signaling Technology, Danvers, MA) anti-Rac1. Immunoreactive bands, visualized by enhanced chemiluminescence, were analyzed using a FluorChem HD2 gel analyzer (Alpha Innotech, San Leandro, CA). Band density readings were used to generate a ratio of active to total Rac1. Ratios were subjected to Kruskal-Wallis analysis of variance (ANOVA) and Mann-Whitney U post hoc analyses at = 0.05 (SPSS, Chicago, IL). RhoA activation in growth cones and whole cell lysates was measured using the Rho G-LISA assay (Cytoskeleton, Denver, CO). Following treatment, cells were fractionated and lysed. Samples (30C50 g total protein) were loaded onto a rhotekin coated microtiter plate. Plates were incubated with anti-RhoA primary and HRP-conjugated secondary antibodies (supplied with AZD7507 kit). An HRP detection reagent was added and the absorbance of each well was read at 490 nm. Absorbance readings were blank corrected and normalized by input protein concentration. Positive controls (RhoA-Q63L) were included in each assay. Maximal (A490 = 1.19) and minimal AZD7507 (A490 = 0.26) absorbance readings for RhoA activation were obtained from GTP and GDP loaded RhoA protein, respectively. Data were analyzed using Kruskal-Wallis ANOVA and Mann-Whitney U post hoc analyses with a significance level of = 0.05. The antibody 2G13 labeled both growth cone regions and the putative growth cone fraction in B35 cells. Growth cone regions, identified from phase contrast images (Fig. 1A), were highly 2G13 immunoreactive (Fig. 1B). Primary omitted control samples confirmed labeling specificity (data not shown). In immunoblots from fractionated cells, the 37 kDa 2G13p was present primarily in the putative growth cone fraction (Fig. 1C) . Sister Coomassie blue-stained gels showed abundant protein in both fractions (Fig. 1D). Because total protein appeared to be less in the pellet, we estimated the immunoreactivity in the putative growth cone fraction by normalizing densities for immunoreactive bands in each fraction to the sum of Coomassie blue-stained band densities in each fraction (excluding major histones, bottom of the pellet lane). These calculations revealed a more than 11 fold increase in band density for 2G13p in the growth cone fraction compared with the AZD7507 cell body fraction. Additionally, immunoreactivity for the nuclear marker lamin B was evident only in the pellet fraction (Fig. 1E). Western blotting for actin confirmed its abundance in growth cone fractions (Fig. 1F),.
Additionally, 8-Br-cAMP reversed increases in RhoA activity induced by Sema 3A in whole cell lysates and CSPGs in growth cones