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Santa Cruz Biotechnology rabbit polyclonal antibody against rac1
Rabbit Polyclonal Antibody Against Rac1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 95 stars, based on 1251 article reviews

rabbit polyclonal antibody against rac1 - by Bioz Stars, 2026-07

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Overall survival and the expression levels of Rho/Rac family genes in DLBCL. Kaplan–Meier curve of overall survival for individual member ( A ) RAC1, ( B ) RAC2, ( C ) RND1, ( D ) CDC42, ( E ) RHOQ, ( F ) RHOF; HR, hazard ratio.

Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: Overall survival and the expression levels of Rho/Rac family genes in DLBCL. Kaplan–Meier curve of overall survival for individual member ( A ) RAC1, ( B ) RAC2, ( C ) RND1, ( D ) CDC42, ( E ) RHOQ, ( F ) RHOF; HR, hazard ratio.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques: Expressing

Correlation between the expression of Rho/Rac family members’ and BTK. ( A ) Co-expression heatmap of Rho/Rac family genes and BTK; ( B ) Scatter diagram demonstrated the correlation between BTK and RAC1 , ( C ) CDC42, ( D ) RHOF and ( E ) RHOQ. * p <0.05; ** p < 0.01; *** p < 0.001.

Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: Correlation between the expression of Rho/Rac family members’ and BTK. ( A ) Co-expression heatmap of Rho/Rac family genes and BTK; ( B ) Scatter diagram demonstrated the correlation between BTK and RAC1 , ( C ) CDC42, ( D ) RHOF and ( E ) RHOQ. * p <0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques: Expressing

RAC1 expression levels in 18 different types of tumor tissues and paired adjacent normal tissues. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: non-significance. BLCA, Bladder Urothelial Carcinoma; BRCA, Breast invasive carcinoma; CHOL, Cholangio carcinoma; COAD, Colon adenocarcinoma; ESCA, Esophageal carcinoma; HNSC, Head and Neck squamous cell carcinoma; KICH, Kidney Chromophobe; KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma; PAAD, Pancreatic adenocarcinoma; PRAD, Prostate adenocarcinoma; READ, Rectum adenocarcinoma; STAD, Stomach adenocarcinoma; THCA, Thyroid carcinoma; UCEC, Uterine Corpus Endometrial Carcinoma.

Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: RAC1 expression levels in 18 different types of tumor tissues and paired adjacent normal tissues. * p < 0.05; ** p < 0.01; *** p < 0.001; ns: non-significance. BLCA, Bladder Urothelial Carcinoma; BRCA, Breast invasive carcinoma; CHOL, Cholangio carcinoma; COAD, Colon adenocarcinoma; ESCA, Esophageal carcinoma; HNSC, Head and Neck squamous cell carcinoma; KICH, Kidney Chromophobe; KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma; PAAD, Pancreatic adenocarcinoma; PRAD, Prostate adenocarcinoma; READ, Rectum adenocarcinoma; STAD, Stomach adenocarcinoma; THCA, Thyroid carcinoma; UCEC, Uterine Corpus Endometrial Carcinoma.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques: Expressing

Correlation between RAC1 expression and DLBCL clinical characteristics.

Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: Correlation between RAC1 expression and DLBCL clinical characteristics.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques: Expressing

Logistic analyses of RAC1 expression and clinical characteristics of DLBCL.

Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: Logistic analyses of RAC1 expression and clinical characteristics of DLBCL.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques: Expressing

RAC1 expression in patients with DLBCL according to different clinical stages.( A ) In the TCGA-DLBC dataset, RAC1 was upregulated in stage I/II as compared with that in stage III/IV. ( B ) RAC1 protein IHC staining in lymphadenitis and ( C ) DLBCL stage II, ( D ) stage III, ( E ) stage IV. Magnification 20 × 20, * p < 0.05. The staining of 3,3′-diaminobenzidine (DAB) was employed for RAC1 expression (yellow and brown) compared to negative sample (blue). Scale bar (bottom, right) = 100 μm.

Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: RAC1 expression in patients with DLBCL according to different clinical stages.( A ) In the TCGA-DLBC dataset, RAC1 was upregulated in stage I/II as compared with that in stage III/IV. ( B ) RAC1 protein IHC staining in lymphadenitis and ( C ) DLBCL stage II, ( D ) stage III, ( E ) stage IV. Magnification 20 × 20, * p < 0.05. The staining of 3,3′-diaminobenzidine (DAB) was employed for RAC1 expression (yellow and brown) compared to negative sample (blue). Scale bar (bottom, right) = 100 μm.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques: Expressing, Immunohistochemistry, Staining

Cox regression analyses of clinical features associated with DLBCL overall survival.

Journal: Cells

Article Title: RAC1 , a Potential Diagnostic and Prognostic Marker for Diffuse Large B Cell Lymphoma

doi: 10.3390/cells11244039

Figure Lengend Snippet: Cox regression analyses of clinical features associated with DLBCL overall survival.

Article Snippet: Sections from paraffin-embedded tissues were then immunostained with a rabbit polyclonal antibody against RAC1 (dilution: 1:300, Bioss, BJ, CHN).

Techniques:

The correlation of integrin αvβ6 expression and RAC1 expression with clinicopathologic variables in cases of gastric cancer.

Journal: Frontiers in Oncology

Article Title: The association and clinicopathological significance of Integrin alphavbeta6 and Rac1 expression in gastric carcinoma

doi: 10.3389/fonc.2024.1347270

Figure Lengend Snippet: The correlation of integrin αvβ6 expression and RAC1 expression with clinicopathologic variables in cases of gastric cancer.

Article Snippet: Rabbit polyclonal antibody against ITGB6 (dilution, 1:500; #28378-1-AP; Proteintech; USA) and Rabbit polyclonal antibody against Rac1 (dilution, 1:500; #24072-1-AP; Proteintech; USA) were utilized.

Techniques: Expressing

The manifestation of ITGB6 and Rac1 in diverse specimens. (A) Immunohistochemical staining exhibited diminished ITGB6 expression in adjacent normal tissues. (B) Immunohistochemical staining revealed elevated ITGB6 expression in tumor tissues. (C) ITGB6 expression in tumor tissues lacking lymphatic invasion. (D) ITGB6 expression in tumor tissues with lymphatic invasion. (E) Immunohistochemical staining displayed reduced Rac1 expression in adjacent normal tissues. (F) Immunohistochemical staining exhibited heightened Rac1 expression in tumor tissues. (G) Rac1 expression in tumor tissues without lymphatic invasion. (H) Rac1 expression in tumor tissues with lymphatic invasion.

Journal: Frontiers in Oncology

Article Title: The association and clinicopathological significance of Integrin alphavbeta6 and Rac1 expression in gastric carcinoma

doi: 10.3389/fonc.2024.1347270

Figure Lengend Snippet: The manifestation of ITGB6 and Rac1 in diverse specimens. (A) Immunohistochemical staining exhibited diminished ITGB6 expression in adjacent normal tissues. (B) Immunohistochemical staining revealed elevated ITGB6 expression in tumor tissues. (C) ITGB6 expression in tumor tissues lacking lymphatic invasion. (D) ITGB6 expression in tumor tissues with lymphatic invasion. (E) Immunohistochemical staining displayed reduced Rac1 expression in adjacent normal tissues. (F) Immunohistochemical staining exhibited heightened Rac1 expression in tumor tissues. (G) Rac1 expression in tumor tissues without lymphatic invasion. (H) Rac1 expression in tumor tissues with lymphatic invasion.

Techniques: Immunohistochemical staining, Staining, Expressing

(A) The level of ITGB6 expression was assessed using the H-score in both adjacent normal tissue specimens and tumor tissues. (B) The H-score of ITGB6 expression was evaluated in adjacent normal tissue specimens, comparing those with lymph node metastasis and those without. (C) We measured the H-score of Rac1 expression in both adjacent normal tissue specimens and tumor tissues. (D) The H-score of Rac1 expression was analyzed in adjacent normal tissue specimens, comparing those with lymph node metastasis and those without. *P<0.05; **P<0.001; ***P<0.0001.

Journal: Frontiers in Oncology

Article Title: The association and clinicopathological significance of Integrin alphavbeta6 and Rac1 expression in gastric carcinoma

doi: 10.3389/fonc.2024.1347270

Figure Lengend Snippet: (A) The level of ITGB6 expression was assessed using the H-score in both adjacent normal tissue specimens and tumor tissues. (B) The H-score of ITGB6 expression was evaluated in adjacent normal tissue specimens, comparing those with lymph node metastasis and those without. (C) We measured the H-score of Rac1 expression in both adjacent normal tissue specimens and tumor tissues. (D) The H-score of Rac1 expression was analyzed in adjacent normal tissue specimens, comparing those with lymph node metastasis and those without. *P<0.05; **P<0.001; ***P<0.0001.

Techniques: Expressing

(A) Kaplan-Meier survival curves based on the ITGB6 gene expression level. (B) Kaplan-Meier survival curves based on the Rac1 gene expression level. (C) Receiver Operating Characteristic (ROC) analysis was conducted to predict the prognosis of gastric cancer patients using ITGB6 and Rac1 expression. (D) Receiver Operating Characteristic (ROC) analysis was conducted to predict lymph node metastasis in gastric cancer patients using ITGB6 and Rac1 expression.

Journal: Frontiers in Oncology

Article Title: The association and clinicopathological significance of Integrin alphavbeta6 and Rac1 expression in gastric carcinoma

doi: 10.3389/fonc.2024.1347270

Figure Lengend Snippet: (A) Kaplan-Meier survival curves based on the ITGB6 gene expression level. (B) Kaplan-Meier survival curves based on the Rac1 gene expression level. (C) Receiver Operating Characteristic (ROC) analysis was conducted to predict the prognosis of gastric cancer patients using ITGB6 and Rac1 expression. (D) Receiver Operating Characteristic (ROC) analysis was conducted to predict lymph node metastasis in gastric cancer patients using ITGB6 and Rac1 expression.

Techniques: Gene Expression, Expressing

The correlation between the expression of ITGB6 and Rac1 in human gastric tumor tissues (r =0.285, P <0.001).

Journal: Frontiers in Oncology

Article Title: The association and clinicopathological significance of Integrin alphavbeta6 and Rac1 expression in gastric carcinoma

doi: 10.3389/fonc.2024.1347270

Figure Lengend Snippet: The correlation between the expression of ITGB6 and Rac1 in human gastric tumor tissues (r =0.285, P <0.001).

Techniques: Expressing

(A) Survival analysis was conducted on gastric cancer patients in a retrospective cohort, taking into consideration the combined levels of ITGB6 and Rac1. Based on the expression levels of ITGB6 and Rac1, the patients were classified into four groups, and subsequently, survival rates were calculated and represented using Kaplan-Meier curves. (B) In order to predict the prognosis of gastric cancer patients, a Receiver Operating Characteristic (ROC) analysis was performed, utilizing the combined expression levels of ITGB6 and Rac1.

Journal: Frontiers in Oncology

Article Title: The association and clinicopathological significance of Integrin alphavbeta6 and Rac1 expression in gastric carcinoma

doi: 10.3389/fonc.2024.1347270

Figure Lengend Snippet: (A) Survival analysis was conducted on gastric cancer patients in a retrospective cohort, taking into consideration the combined levels of ITGB6 and Rac1. Based on the expression levels of ITGB6 and Rac1, the patients were classified into four groups, and subsequently, survival rates were calculated and represented using Kaplan-Meier curves. (B) In order to predict the prognosis of gastric cancer patients, a Receiver Operating Characteristic (ROC) analysis was performed, utilizing the combined expression levels of ITGB6 and Rac1.

Techniques: Expressing

Univariate and multivariate analyses applying the Cox proportional hazard model to patients diagnosed with gastric cancer (Retrospective cohort).

Journal: Frontiers in Oncology

Article Title: The association and clinicopathological significance of Integrin alphavbeta6 and Rac1 expression in gastric carcinoma

doi: 10.3389/fonc.2024.1347270

Figure Lengend Snippet: Univariate and multivariate analyses applying the Cox proportional hazard model to patients diagnosed with gastric cancer (Retrospective cohort).

Techniques: Significance Assay

(A) Transfection of ITGB6 siRNA was conducted in the SGC7901 gastric cancer cell line, and the transfection efficiency was evaluated using RT-PCR. (B) After inhibiting the expression of ITGB6 in 7901 cells and treating them with the Rac1 activity inhibitor NSC23766, the cell viability was assessed using the CCK8 assays. (C) After interfering with ITGB6 expression in 7901 cells and treating them with the Rac1 activity inhibitor NSC23766, Transwell migration and invasion assays were performed to assess the changes in cell migration and invasion abilities. (D) Transfection of NC and ITGB6 siRNA was performed separately in 7901 cells, followed by treatment with NSC23766. Cell viability was evaluated using the CCK8 assay. (E) In the experiment, we performed co-transfection of NC and ITGB6 siRNA in 7901 cells, accompanied by NSC23766 treatment. The migration and invasion abilities of the cells were assessed through Transwell migration and invasion assays. *P<0.05; **P<0.001; ***P<0.0001

Journal: Frontiers in Oncology

Article Title: The association and clinicopathological significance of Integrin alphavbeta6 and Rac1 expression in gastric carcinoma

doi: 10.3389/fonc.2024.1347270

Figure Lengend Snippet: (A) Transfection of ITGB6 siRNA was conducted in the SGC7901 gastric cancer cell line, and the transfection efficiency was evaluated using RT-PCR. (B) After inhibiting the expression of ITGB6 in 7901 cells and treating them with the Rac1 activity inhibitor NSC23766, the cell viability was assessed using the CCK8 assays. (C) After interfering with ITGB6 expression in 7901 cells and treating them with the Rac1 activity inhibitor NSC23766, Transwell migration and invasion assays were performed to assess the changes in cell migration and invasion abilities. (D) Transfection of NC and ITGB6 siRNA was performed separately in 7901 cells, followed by treatment with NSC23766. Cell viability was evaluated using the CCK8 assay. (E) In the experiment, we performed co-transfection of NC and ITGB6 siRNA in 7901 cells, accompanied by NSC23766 treatment. The migration and invasion abilities of the cells were assessed through Transwell migration and invasion assays. *P<0.05; **P<0.001; ***P<0.0001

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Activity Assay, Migration, CCK-8 Assay, Cotransfection

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.

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

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.

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

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.

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.

Techniques: Activation Assay, Western Blot

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).

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).

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

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.

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.

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

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).

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).

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

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.

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.

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

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.

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.

Techniques: Plasmid Preparation, Transfection, Control, Staining

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.

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.

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

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.

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.

Techniques: Expressing, Plasmid Preparation, Control, Western Blot

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