affinity purified rabbit polyclonal antibody against rac1 p21  (Santa Cruz Biotechnology)

Journal: Human reproduction (Oxford, England)

Article Title: WAVE1, an A-kinase anchoring protein, during mammalian spermatogenesis.

doi: 10.1093/humrep/deh513

Figure Lengend Snippet: Figure 6. WAVE1, PKA RII and RAC1 distribution in mature sperm. Mature mouse, bull, baboon and human semen samples were studied by immunocytochemistry. (A) A bright signal of WAVE1 (green) can be seen on the mitochondrial sheaths in the majority of mature mouse sperm. (B) PKA RII (red) is visualized as a bright signal in the mitochondrial sheath and, to a lesser extent, also in the principal piece. (C) The small GTPase RAC1 (red) is observed in both equatorial and post-acrosomal regions of the sperm head. A faint staining of RAC1 is also observed on the mitochondrial sheath. In mature bull sperm, a consistent co-localization of WAVE1 and PKA RII is observed on the mitochondrial sheath (A0 and B0). Similar to the mature mouse sperm, RAC1 is preferentially distributed in the equatorial and post-acrosomal regions (C0). Similar co-localization of WAVE1 and PKA RII is detected in mature baboon and human sperm (A00, B00, A000, B000). RAC1 also localizes to the equatorial and post-acrosomal regions in baboon and human sperm. RAC1 co-localizes with WAVE1 in the mitochondrial sheath (insets in C00 and C000). A, A0 and A000 show tubulin in red. Insets in A, A0, A00 and A000 show negative controls after the omission of the primary antibody. Insets in C, C0, C00 and C000 show RAC1 (red) and WAVE1 (green). Bar ¼ 10 mm.

Article Snippet: Antibodies and live/dead sperm staining Affinity-purified goat polyclonal antibody, raised against a peptide mapping near the carboxy terminus of WAVE1 (human origin), and affinity-purified rabbit polyclonal antibody against RAC1 p21 were obtained from Santa Cruz Biotechnology (USA).

Techniques: Immunocytochemistry, Staining

Journal: Journal of Biological Chemistry

Article Title: The Requirement for Polyamines for Intestinal Epithelial Cell Migration Is Mediated through Rac1

doi: 10.1074/jbc.m208741200

Figure Lengend Snippet: FIG. 1. Relative amounts of total and active (GTP-bound) Rac1, RhoA, and Cdc42 proteins in confluent IEC-6 cells grown for 4 days in control medium (DMEM-containing serum). Cell extracts equivalent to 25–200 g of total protein were subjected to pull-down assays using GST-PAK or GST-PKN. Samples from the pull down assays were resolved by SDS-PAGE along with 25 g of total protein from the same cell extracts. Western blot analysis was performed using RhoA-, Rac1-, and Cdc42-specific antibodies to determine the levels of activated and total Rac1, RhoA, and Cdc42 proteins. Representative Western blots from three observations are shown.

Article Snippet: The primary antibodies, affinitypurified mouse monoclonal antibodies against RhoA and affinity-purified rabbit polyclonal antibodies against Rac1 and Cdc-42, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Control, SDS Page, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Requirement for Polyamines for Intestinal Epithelial Cell Migration Is Mediated through Rac1

doi: 10.1074/jbc.m208741200

Figure Lengend Snippet: FIG. 2. Polyamine depletion inhibits activation of RhoA, Rac1, and Cdc42 in IEC-6 cells. Cells were grown in the presence of 5 mM DFMO or DFMO plus 10 M putrescine for 4 days. Equal amounts of protein (200 g for RhoA and Cdc42 and 100 g for Rac1) were used for the pull-down assays as described under “Experimental Procedures.” 25 g of protein was used to determine the levels of total RhoA, Rac1, and Cdc42 proteins. Western blot (WB) analysis using RhoA-, Rac1-, and Cdc42-specific antibodies was carried out. A, active RhoA protein bound to GST-mDia. B, active RhoA protein bound to GST-PKN. C, RhoA protein levels in whole cell extract. D, active Rac1 protein bound to GST-PAK. E, Rac1 protein levels in whole cell extract. F, active Cdc42 protein bound to GST-PAK. G, Cdc42 protein levels in whole cell ex- tract. H, actin levels in whole cell extract. Representative Western blots and densitometry readings from three observations are shown.

Article Snippet: The primary antibodies, affinitypurified mouse monoclonal antibodies against RhoA and affinity-purified rabbit polyclonal antibodies against Rac1 and Cdc-42, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Activation Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Requirement for Polyamines for Intestinal Epithelial Cell Migration Is Mediated through Rac1

doi: 10.1074/jbc.m208741200

Figure Lengend Snippet: FIG. 3. Time course of the effect of polyamine depletion on the activation of RhoA, Rac1, and Cdc42 in IEC-6 cells. A control group of cells was grown for 4 days in DMEM/FBS to confluence. 5 mM DFMO was added to other groups on days 0, 1, 2, or 3 during the 4-day growth period to deplete polyamines for 4, 3, 2, and 1 days respectively. Another group was exposed to DFMO plus 10 M putrescine (PUT) for 4 days. A, 200 g (for RhoA, Cdc42) and 100 g (for Rac1) of total protein from each sample was used for the pull-down assay using GST-PKN or GST-PAK fusion protein. GST-PKN- or GST-PAK-bound proteins were resolved by SDS-PAGE and subjected to Western blot analysis using antibodies specific for RhoA, Rac1, and Cdc42. 25 g of protein from whole cell extracts was resolved by SDS-PAGE and sub- jected to Western blot analysis for the detection of total RhoA, Rac1, and Cdc42 protein. Representative Western blots from 6 observations are shown. B, specific activities of RhoA, Rac1, and Cdc42 calculated by the densitometric readings of the Western blots. Values are means S.E. of six observations. *, significantly different from control (p 0.05).

Article Snippet: The primary antibodies, affinitypurified mouse monoclonal antibodies against RhoA and affinity-purified rabbit polyclonal antibodies against Rac1 and Cdc-42, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Activation Assay, Control, Pull Down Assay, SDS Page, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Requirement for Polyamines for Intestinal Epithelial Cell Migration Is Mediated through Rac1

doi: 10.1074/jbc.m208741200

Figure Lengend Snippet: FIG. 4. IEC-6 cells transfected with vector (pMX-IRES-GFP), dominant negative (DN) Rac1, and Cdc42 (N17-Rac1, N17- Cdc42) and constitutively active (CA) Rac1 and Cdc42 (V12- Rac1, F28L-Cdc42) were grown for 4 days as described earlier and in 35-mm dishes (each containing a matrigel-coated glass coverslip). Expression of green fluorescence protein was detected by digital confocal microscopy, and the images were processed with NIH image.

Article Snippet: The primary antibodies, affinitypurified mouse monoclonal antibodies against RhoA and affinity-purified rabbit polyclonal antibodies against Rac1 and Cdc-42, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Transfection, Plasmid Preparation, Dominant Negative Mutation, Expressing, Fluorescence, Confocal Microscopy

Journal: Journal of Biological Chemistry

Article Title: The Requirement for Polyamines for Intestinal Epithelial Cell Migration Is Mediated through Rac1

doi: 10.1074/jbc.m208741200

Figure Lengend Snippet: FIG. 5. Rac1 and Cdc42 protein expression influences IEC-6 cell migration. A, cells transfected with vector (pMX-IRES-GFP), con- stitutively active (CA) Rac1 and Cdc42, and dominant negative (DN) Rac1 and Cdc42 were grown in DMEM, 5% FBS for 4 days. Confluent monolayers were wounded with a gel-loading tip in the center of plates marked to localize the wound site. Plates were photographed immedi- ately to record the wound width (0 h), washed, and incubated with fresh serum free medium. Plates were photographed at the marked wound location after 10 h of incubation. A plate containing vector cells at 0 and 10 h is shown as a representative control. Representatives of three experiments are shown. B, quantitative analysis of migration showing wound width covered as compared with initial scratch size (0 h) using NIH image analysis. Values are the mean S.E. of six observations. *, Significantly different from vector (p 0.05). DN, dominant negative; CA, constitutively active. FIG. 6. Activated recombinant Rac1 and Cdc42 expression in- fluences the migration of polyamine-depleted IEC-6 cells. A, vector (pMX-IRES-GFP) and constitutively active V12-Rac1- and F28L- Cdc42-transfected cells were grown in control media, control media plus 5 mM DFMO, and control media plus DFMO plus 10 M putrescine for 4 days. After serum starvation, monolayers were wounded as described in Fig. 5. 0- and 8-h images of wounds are shown for control, DFMO and DFMO putrescine (Put) group from the same plate. Panels are rep- resentative of six observations. B, quantitative analysis of migration showing wound width covered (%) compared with initial scratch size (0 h) using NIH image analysis. Values are the mean S.E. of six observations. *, Significantly different from control (p 0.05).

Article Snippet: The primary antibodies, affinitypurified mouse monoclonal antibodies against RhoA and affinity-purified rabbit polyclonal antibodies against Rac1 and Cdc-42, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Expressing, Migration, Transfection, Plasmid Preparation, Dominant Negative Mutation, Incubation, Control, Recombinant

Journal: Journal of Biological Chemistry

Article Title: The Requirement for Polyamines for Intestinal Epithelial Cell Migration Is Mediated through Rac1

doi: 10.1074/jbc.m208741200

Figure Lengend Snippet: FIG. 7. Polyamine depletion does not alter Rac1 and Cdc42 activation in constitutively active (V12) Rac and (F28L) Cdc42 cells but inhibits Rac1 activation in vector (pMX-IRES-GFP)- transfected IEC-6 cells. Extracts of control cells (C) and those grown in DFMO (D) or DFMO plus putrescine (DP) for 4 days were subjected to the GST-PAK pull down assay described “Experimental Procedures.” Extracts of vector-transfected cells were also preincubated with GTPS for 15 min before the addition of GST-PAK for the pull-down assay. 25 g of total protein was used to determine total Rac1 and Cdc42 protein. Western blot analysis was performed using Rac1- and Cdc42-specific antibodies to determine the levels of activated and total Rac1 and Cdc42 proteins. Representative Western blots and densitometry readings from three observations are shown.

Article Snippet: The primary antibodies, affinitypurified mouse monoclonal antibodies against RhoA and affinity-purified rabbit polyclonal antibodies against Rac1 and Cdc-42, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Activation Assay, Plasmid Preparation, Transfection, Control, Pull Down Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Requirement for Polyamines for Intestinal Epithelial Cell Migration Is Mediated through Rac1

doi: 10.1074/jbc.m208741200

Figure Lengend Snippet: FIG. 8. Constitutively active Rac1 prevents the effects of polyamine de- pletion on the F-actin cytoskeleton of IEC-6 cells. Vector (pMX-IRES-GFP) and constitutively active (V12) Rac1- transfected cells were grown for 4 days as described earlier and replated in 35-mm dishes (each containing a matrigel-coated glass coverslip). Cells were allowed to at- tach and spread for 24 h in control and DFMO- and DFMO plus putrescine (Put)- containing medium. Cells were washed, fixed, and stained with Texas Red phal- loidin for the localization of F-actin. Rep- resentatives of three experiments are shown.

Article Snippet: The primary antibodies, affinitypurified mouse monoclonal antibodies against RhoA and affinity-purified rabbit polyclonal antibodies against Rac1 and Cdc-42, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Plasmid Preparation, Transfection, Control, Staining

Journal: Journal of Biological Chemistry

Article Title: The Requirement for Polyamines for Intestinal Epithelial Cell Migration Is Mediated through Rac1

doi: 10.1074/jbc.m208741200

Figure Lengend Snippet: FIG. 9. Constitutively active V12-Rac1 activates RhoA and Cdc42 in polyamine-depleted IEC-6 cells. Extracts of vector and V12-Rac1 transfected-cells grown as control (C), DFMO (D), or DFMO plus putrescine (DP) groups for 4 days were subjected to the GST-PAK and GST-PKN pull-down assay described in the under “Experimental Procedures.” Western blot analysis was performed using RhoA-, Rac1-, and Cdc42-specific antibodies to determine the levels of activated Rac1, RhoA, and Cdc42 proteins as well as total protein. Representative Western blots from three observations are shown.

Article Snippet: The primary antibodies, affinitypurified mouse monoclonal antibodies against RhoA and affinity-purified rabbit polyclonal antibodies against Rac1 and Cdc-42, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Plasmid Preparation, Transfection, Control, Pull Down Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Requirement for Polyamines for Intestinal Epithelial Cell Migration Is Mediated through Rac1

doi: 10.1074/jbc.m208741200

Figure Lengend Snippet: FIG. 10. Activities of Rho GTPases in normal and polyamine-depleted cells expressing constitutively active RhoA and Cdc42. Vector and V14-RhoA- and F28L-Cdc42-ransfected cells were grown for 4 days in control medium (C) and control medium with DFMO (D) and DFMO plus putrescine (DP). Cell extracts were used for pull-down assays for RhoA, Rac1, and Cdc42 as described in Fig. 9. Western blot analysis was performed using RhoA-, Rac1-, and Cdc42-specific antibodies to determine the levels of activated Rac1, RhoA, and Cdc42 proteins. Representative Western blots from three observations are shown.

Article Snippet: The primary antibodies, affinitypurified mouse monoclonal antibodies against RhoA and affinity-purified rabbit polyclonal antibodies against Rac1 and Cdc-42, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Expressing, Plasmid Preparation, Control, Western Blot