Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: UPARANT is an effective antiangiogenic agent in a mouse model of rubeosis iridis

doi: 10.1007/s00109-019-01794-w

Figure Lengend Snippet: List of antibodies

Article Snippet: Anti-CXCR4 , Rabbit , 1:200 , WB , Bio-Techne Corp., Abingdon, UK , NB100-56437.

Techniques:

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: UPARANT is an effective antiangiogenic agent in a mouse model of rubeosis iridis

doi: 10.1007/s00109-019-01794-w

Figure Lengend Snippet: List of primer-pairs

Article Snippet: Anti-CXCR4 , Rabbit , 1:200 , WB , Bio-Techne Corp., Abingdon, UK , NB100-56437.

Techniques:

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: UPARANT is an effective antiangiogenic agent in a mouse model of rubeosis iridis

doi: 10.1007/s00109-019-01794-w

Figure Lengend Snippet: UPARANT counteracts inflammation and extracellular matrix degradation in iris neovascularization. a Transcript levels of genes involved in hypoxia response, canonical angiogenesis, ECM degradation, and inflammation were quantified by qPCR on non-punctured control (CTRL), RI-induced, and 7.6 g/L UPARANT (UPR) intravitreally treated eyes. Data are presented as box plots of independent eyes per group ( n = 4), and normalized to CTRL. b Representative western blots of VEGF, MMP2, IL-6, and CXCR4, and the loading control actin for CTRL eyes, RI-induced eyes, and UPR-treated eyes by intravitreal administration. Densitometry analysis of protein levels is corrected versus the loading control and results are presented normalized to CTRL as box plots of independent eyes per group ( n = 4). Statistical analysis was performed by one-way ANOVA with Bonferroni posttest ( p < 0.05: * vs. CTRL, ° vs. RI)

Article Snippet: Anti-CXCR4 , Rabbit , 1:200 , WB , Bio-Techne Corp., Abingdon, UK , NB100-56437.

Techniques: Western Blot

Journal: STAR Protocols

Article Title: Protocol for generation and engineering of thyroid cell lineages using CRISPR-Cas9 editing to recapitulate thyroid cancer histotype progression

doi: 10.1016/j.xpro.2024.103263

Figure Lengend Snippet:

Article Snippet: Resuspend 1·10 5 cells in a volume of 100 μL were exposed for 1 h at 4°C to specific markers : Oct3/4 (40/Oct-3 Alexa-Fluor 647, mouse IgG1K, BD Biosciences, 20 μL/sample), Sox2 (245610 PE, mouse IgG2a BD Biosciences, 20 μL/sample), Nanog (N31-355 PE, mouse IgG1, BD Biosciences, 20 μL/sample), CXCR4 (FAB170P PE, mouse IgG2a, R&D system, 1 μL/sample), c-kit (YB5.B8 PE, mouse IgG1 BD Biosciences, 20 μL/sample), Sox17 (P7-969 PE, mouse IgG1k, BD Biosciences, 5 μL/sample), Fox A2 (N17-280 PE, mouse IgG1, BD Biosciences 5 μL/sample), PAX8 (PAX8/1492 APC, mouse IGg2a, Novus 5 μL/sample), TTF1 (REA1090 FITC, mouse IgG1, MACS Miltenyi Biotec, 16 μL/sample), TSH-R (4C1 FITC, IgG2a Santa Cruz, 2 μL/sample), TPO (203340, Anti-rabbit IgG H + L Alexa-488, Abcam, 2 μL/sample), Thyroglobulin (SPM221 PE, mouse IgG1, Novus, 2 μL/sample), NIS (SPM186, goat anti-mouse IgG (H + L) Alexa-488, Abcam, 5 μL/sample), CD133 (W6B3C1 FITC, mouse IgG1, BD Bioscience, 20 μL/sample), ABCG2 (5D3/CD338 APC, mouse IgG2b, BD Bioscience, 10 μL/sample), Nestin (25/Nestin PerCP 5.5, mouse IgG1, BD Bioscience, 5 μL/sample), HNF-4α (H-1, goat anti-mouse IgG (H + L) Alexa-488, Santa Cruz, 4 μL/sample), or corresponding isotype matched controls (IMC). xiii.

Techniques: Recombinant, Transfection, Reverse Transcription, Purification, Sequencing, Plasmid Preparation, CRISPR, Software, Cell Culture, Sterility

Journal: Stem Cell Reviews and Reports

Article Title: Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts

doi: 10.1007/s12015-020-10005-w

Figure Lengend Snippet: Confocal analysis of membrane lipid rafts in purified murine SKL cells . Panel A. Defective lipid raft formation in murine C57Bl/6 Nlrp3-KO BM-purified SKL cells or wild type BM-purified SKL cells exposed to adenosine (10 μM) or CoPP (50 μM). Representative images of SKL cells sorted from WT BM, stimulated with SDF-1 (50 ng/ml) and LL-37 (2.5 μg/ml), stained with cholera toxin subunit B (a lipid raft marker) conjugated with FITC and rat anti-mouse CXCR4 followed by anti-rat Alexa Fluor 594, and evaluated by confocal microscopy for formation of membrane lipid rafts. Lipid rafts were formed in SKL cells (control) but not in SKL cells isolated from Nlrp3-KO animals or SKL cells isolated from WT animals after adenosine and CoPP treatment. Panel B. The chemotactic responsiveness of mBMMNCs untreated or treated with 10 Panx, SC Panx, or apyrase in unsupplemented medium or medium supplemented with SDF-1, S1P, or ATP, as determined by counting the number of CFU-GM clonogenic progenitors. Results are combined from two independent experiments. *p > 0.05, ** p > 0.01, *** p > 0.001

Article Snippet: The cholera toxin B subunit conjugated with FITC (Sigma-Aldrich) was applied to detect the ganglioside GM1, and rat monoclonal anti-CXCR4 IgG antibody (R&D Systems) and Alexa Fluor 594 goat anti-rat IgG antibody (Invitrogen) were applied to detect CXCR4.

Techniques: Membrane, Purification, Staining, Marker, Confocal Microscopy, Isolation

Journal: Stem Cell Reviews and Reports

Article Title: Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts

doi: 10.1007/s12015-020-10005-w

Figure Lengend Snippet: The impact of pannexin 1 channel blockade on short- and long-term engraftment of HSPCs in WT mice. Panel A. Lethally irradiated WT mice (9 per group) were transplanted with bone marrow mononuclear cells (BMMNCs) that had been previously labeled with a PKH67 cell linker and then treated with 10 Panx or SC Panx. Twenty-four hours after transplantation, the femoral BMMNCs were harvested, the number of PKH67 + cells evaluated by FACS, and the CFU-GM clonogenic progenitors enumerated in an in vitro colony assay. Panel B. Lethally irradiated WT mice (9 per group) were transplanted with BMMNCs treated with 10 Panx or SC Panx, and 12 days after transplantation the femoral BMMNCs were harvested and plated to count the number of CFU-GM colonies and the spleens removed for counting the number of CFU-S colonies. No colonies were formed in lethally irradiated, untransplanted mice (irradiation control). * p < 0.05, ** p < 0.01, *** p < 0.001. Panel C. Lethally irradiated WT mice (9 per group) were transplanted with BMMNCs treated with 10 Panx. White blood cells (above) and platelets (below) were counted at intervals (at 0, 3, 7, 14, 21, and 28 days after transplantation). * p < 0.05. Panel D . Defective lipid raft formation in murine BM-purified SKL cells from C57Bl/6 mice exposed to 10 Panx inhibitory peptide (200 μM). Representative images of SKL cells sorted from WT BM, stimulated with SDF-1 (50 ng/ml) and LL-37 (2.5 μg/ml) (positive control) or exposed to 10 Panx inhibitory peptide, stained with cholera toxin subunit B (a lipid raft marker) conjugated with FITC and rat anti-mouse CXCR4 followed by anti-rat Alexa Fluor 594, and evaluated by confocal microscopy for formation of membrane lipid rafts. Lipid rafts were formed in SKL cells (control), but not in SKL cells after 10 Panx inhibitory peptide treatment

Article Snippet: The cholera toxin B subunit conjugated with FITC (Sigma-Aldrich) was applied to detect the ganglioside GM1, and rat monoclonal anti-CXCR4 IgG antibody (R&D Systems) and Alexa Fluor 594 goat anti-rat IgG antibody (Invitrogen) were applied to detect CXCR4.

Techniques: Irradiation, Labeling, Transplantation Assay, In Vitro, Colony Assay, Purification, Positive Control, Staining, Marker, Confocal Microscopy, Membrane

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: Cells were cultured as described in “ ” and treated with E2 or ICI 184,780 (ICI) using EtOH as vehicle. The vehicle-only treatment served as a control. The mRNA levels of CXCL12 (A and B), CXCR4 (D and E) and CXCR7 (G and H) were quantified by real-time PCR analysis of cells treated for different periods of time with 10 −8 M E2 (A, D and G) or cells treated for 48 h with 10 −8 M E2 alone, 10 −6 M ICI alone or both E2 and ICI (B, E and H). The real-time PCR results were normalized against the internal control GAPDH and expressed as the mean CXCL12 , CXCR4 or CXCR7/GAPDH mRNA ratio ± SEM of at least three independent experiments. Protein levels of CXCL12 (C), CXCR4 (F) and CXCR7 (I) was assayed. Secreted CXCL12 protein levels after treatment with EtOH or 10 −8 M E2 for 48 h were determined by ELISA, and the values were normalized relative to the total protein concentration (C). The expression of CXCR4 and CXCR7 at the surface of MCF-7 cells was measured by flow cytometry after treatment with EtOH or 10 −8 M E2 for 48 h (F, I). Representative data from at least three experiments performed in duplicate are shown. Asterisks or different lowercase letters indicate significant differences ( p <0.05) between the control and treated cells.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Cell Culture, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Protein Concentration, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: CXCL12 , CXCR4 and CXCR7 mRNA were assessed by quantitative real time PCR after 48 h treatment of ZR-75 (A) or MDA-MB-231 (B) cells to EtOH (−) or to10 −8 M E2 (+). Transcript levels were normalized against GAPDH mRNA and data were calculated as percentage of the E2 effect. Data are from triplicate samples and are representative of three separate experiments. Asterisk indicates significant differences ( p <0.05) between the control and ligand treated cells.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: The levels of the CXCL12, CXCR4 and CXCR7 transcripts were assessed by quantitative real-time PCR in MCF-7 cells treated under various conditions for 48 h. Treatment with EtOH and 10 −8 M E2 served as the negative and positive controls, respectively. In each experimental assay, the cells were exposed to different concentrations of 17 α-ethynyl-estradiol (EE2) (A) and Genistein (Gen) (B). Transcript levels were normalized against GAPDH mRNA, and data were calculated as percentage of the E2 effect for each experiment. Significant differences (P<0.05) are indicated by different lowercase letters.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: (A) FAIRE assays were performed on MCF-7 cells exposed to either ETOH (−) or 10 −8 M E2 (+) for 48 h. Real-time PCR was performed to monitor enrichment of the DNA corresponding to the proximal promoters of the CXCL12 , CXCR4 and CXCR7 genes relative to input chromatin. The data are from triplicate samples and are representative of three separate experiments. Asterisks indicate significant differences ( p <0.05) between the control and treated cells. (B) The Integrated Genome Browser (Affymetrix) was used to visualize ER-binding sites in the regions surrounding the CXCL12 , CXCR4 and CXCR7 genes. Raw ChIP-chip data for ER and high confidence ER-binding sites called using the MAT algorithm are shown , . The numbered ER-binding sites correspond to bound regions in which the closest TSS is that of CXCL12 , CXCR4 or CXCR7 . Arrows indicate the orientation of the CXCL12 , CXCR4 and CXCR7 genes.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Real-time Polymerase Chain Reaction, Control, Binding Assay, ChIP-chip

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: siRNA directed against CXCL12, CXCR4 or CXCR7 was transfected into MCF-7 cells treated with EtOH (−) or 10 −8 M E2 (+). (A) After 48 h, the levels of the CXCL12, CXCR4 and CXCR7 transcripts were assessed by quantitative PCR and normalized against GAPDH mRNA. The results were compared with those obtained from MCF-7 cells transfected with a nonspecific siRNA control. (B) Total protein was extracted from MCF-7 cells, and the levels of CXCL12, CXCR4 and CXCR7 were analyzed by Western blotting. (C) To determine the growth rate of the MCF-7 cells, the siRNA-transfected cells were treated with EtOH (−E2) or 10 −8 M E2 (+E2) for seven days. E2-dependent and -independent cell growth were evaluated using MTT assays of three independent experiments (n = 6). The results are expressed as a percentage of the relative cell number obtained from cells transfected with the control siRNA and treated with EtOH (considered as 100%). Significant differences ( p <0.05) between transfected cells in the absence of E2 are indicated by an asterisk and between transfected cells in the presence of E2 by a sharp symbol. (D) The effects of specific inhibitors for CXCL12 (Chalcon 4), CXCR4 (AMD3100) or CXCR7 (CCX771) were measured after treatment of MCF-7 cells with either EtOH (−E2) or 10 −8 M E2 (+E2) for 7 days. DMSO (vehicle) was used as the control. E2-dependent and E2-independent cell growth were then evaluated by MTT assays of three independent experiments (n = 6). The results are expressed as a percentage of the relative cell number obtained from cells treated with the vehicle control (considered as 100%). Significant differences ( p <0.05) between treated cells in the absence of E2 are indicated by an asterisk and between treated cells in the presence of E2 by a sharp symbol.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: MCF-7 cells were transiently transfected with either a control expression vector or one containing the human CXCR7 open reading frame. (A) Total protein extracts were prepared 48 h after transfection, and a Western blot analysis was performed to confirm CXCR7 over-expression. (B) Transfected cells were cultured in the presence of EtOH (−) or 10 −8 M E2 (+) for seven days. E2-dependent and E2-independent cell growth rates were then evaluated by cell count of three independent experiments (n = 3). The results are expressed as a percentage of the relative cell number obtained from control cells treated with E2 (considered as 100%). Significant differences ( p <0.05) between transfected cells in the absence of E2 are indicated by an asterisk and between transfected cells in the presence of E2 by a sharp symbol. (C) A proposed model for the involvement of the CXCL12 signaling axis in E2-dependent and -independent cell growth is shown. The binding of CXCL12 to CXCR4 and CXCR7 leads to the stimulation of cell growth through diverse pathways . CXCR7 can also modulate CXCL12 availability by removing the chemokine from the extracellular space (left panel). Estrogens could stimulate cell growth by favoring the activation of CXCL12 through CXCR4 and reducing the expression of CXCR7 (right panel).

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Western Blot, Over Expression, Cell Culture, Cell Counting, Binding Assay, Activation Assay

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A&B) The effect of IL-1β on the mRNA expression of chemokine receptors in Tca8113 (A) and Hep2 (B) cells. Cells were treated with 20 ng/ml IL-1β for 24 h. The mRNA levels of CXCR4, CCR6 and CCR7 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (C) Quantitative CXCR4 mRNA expression in (A). * P < 0.05 compared with the non-treated group. (D) Time course of CXCR4 mRNA expression in response to IL-1β stimulation. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The mRNA levels of CXCR4 were detected by RT-PCR. β-actin mRNA levels were measured as loading controls. (E) The quantitative data corresponding to (D). * P < 0.05 compared with the non-treated group. (F) The effect of IL-1β on CXCR4 protein expression. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. CXCR4 protein expression was detected by FACS. (G) The effect of IL-1β on SDF-1α-induced cell migration. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. Cell migration in response to medium or 20 ng/ml SDF-1α was measured by the Transwell assay. * P < 0.05 compared with control groups. (I) The transwell assay showed cell migration in response to 20 ng/ml SDF-1α after treatment with the indicated concentrations of IL-1β for 24 h (Scale bars: 200 μM).

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Migration, Transwell Assay, Control

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) The expression of IL-1 receptors in Tca8113 cells. Cells were treated with the indicated concentrations of IL-1β for 24 h. The mRNA levels of IL-1R1 and IL-1RII were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (B) The expression of IL-1 receptors in Hep2. Cells were treated as described in (A). The mRNA levels of IL-1R1 and IL-1R2 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (C) The effect of IL-1β on IL-1R1 expression. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. The protein levels of IL-1R1 were measured by western blot. β-actin protein levels were measured as loading controls. (D&E) The effect of IL-1Ra on IL-1β-induced CXCR4 mRNA up-regulation. Tca8113 cells, pre-treated with the indicated concentrations of IL-1Ra for 1 h, were treated with or without 20 ng/ml IL-1β for 1 h. The mRNA levels of CXCR4 were measured by RT-PCR (D) and qRT-PCR (E). * P < 0.05 compared with the IL-1β-treated group. (F) The effect of IL-1Ra on IL-1β-induced CXCR4 protein up-regulation. Tca8113 cells, pre-treated with the indicated concentrations of IL-1Ra for 1 h, were treated with or without 20 ng/ml IL-1β for 24 h. The protein expression of CXCR4 was measured by FACS. (G) The effect of RNA interference on the expression of IL-1R1 protein. Tca8113 cells, transfected with non-specific shRNA (Nssi) or with IL-1R1 shRNA (IL-1R1si), were treated with the indicated concentrations of IL-1β for 24 h. The expression of IL-1R1 protein was measured by western blot. β-actin protein levels were measured as loading controls. (H) The effect of IL-1R1 down-regulation on CXCR4 mRNA expression. Non-specific shRNA (Nssi) or IL-1R1 shRNA (IL-1R1si) transfected Tca8113 cells were treated with medium or 20 ng/ml IL-1β for 24. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (I) The quantitative data corresponding to (H). * P < 0.05 compared with the Nssi group.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Transfection, shRNA

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) The effect of IL-1β on mRNA levels of IL-1β and TNF-α. Tca8113 cells were treated with the indicated concentrations of IL-1β for 24 h. The mRNA levels of IL-1β and TNF-α were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (B) IL-1β quantitative mRNA levels in (A). * P < 0.05 compared with the non-treated control. (C) Time-course of IL-1β mRNA expression in response to IL-1β treatment. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (D) IL-1β quantitative mRNA levels in (C). * P < 0.05 compared with the non-treated group. (E) The effect of IL-1Ra on IL-1β-induced IL-1β mRNA expression. Tca8113 cells, pre-treated with the indicated concentrations of IL-1Ra for 1 h, were stimulated with 20 ng/ml IL-1β for 24 h. The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (F) IL-1β quantitative mRNA levels in (E). * P < 0.05 compared with the non-treated group. (G) The sustained effect of IL-1β on the expression of IL-1β and CXCR4. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated number of days. The mRNA levels of IL-1β and CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (H-I) Quantitative data of IL-1β (H) and CXCR4 (I) in (G). * P < 0.05 compared with the non-treated groups.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Expressing

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) Time-dependent activation of Notch by IL-1β. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. Activated Notch NCID fragments were detected by western blot. β-actin protein levels were measured as loading controls. (B) Dose dependent activation of Notch by IL-1β treatment for 1 h. (C) The effect of IL-1β on Hes1 mRNA levels. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The mRNA levels of the Notch1 targeting gene Hes1 were measured by qRT-PCR. * P < 0.05 compared with the control group. (D) The effect of Notch inhibition on CXCR4 expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of Notch inhibitor L685458 for 30 min, were treated with 20 ng/ml IL-1β for 1 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (E) Quantitative data of CXCR4 expression in (D). * P < 0.05 compared with IL-1β-treated alone group. (F) The effect of Notch inhibition on IL-1β expression induced by IL-1β. Cells were treated as described in (D). The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (G) Quantitative data of IL-1β expression in (F). * P < 0.05 compared with the IL-1β-treated alone group. (H) The effect of Notch inhibition on long-term CXCR4 mRNA expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of Notch inhibitor L685458 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (I) The effect of Notch inhibition on CXCR4 protein expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of the Notch inhibitor L685458 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The protein levels of CXCR4 were measured by western blot. β-actin protein levels were measured as loading controls.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Control, Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

doi: 10.1371/journal.pone.0132677

Figure Lengend Snippet: (A) The effect of IL-1β on the activation of MAPKs. Tca8113 cells were treated with 20 ng/ml IL-1β for the indicated time periods. The phosphorylation levels of MAPKs were measured by western blot. β-actin protein levels were measured as loading controls. (B) The effect of IL-1β on the activation of IκB-α. Tca8113 cells were treated as described in (A). IκB-α protein levels were measured by western blot. β-actin protein levels were measured as loading controls. (C) The effect of ERK inhibition on IL-1β-induced CXCR4 expression. Tca8113 cells, pre-treated with the indicated concentrations of U0126 for 30 min, were stimulated with 20 ng/ml IL-1β for 1 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (D) Quantitative data of (C). * P < 0.05 compared with the IL-1β-treated group. (E) The effect of ERK inhibition on IL-1β-induced IL-1β mRNA expression. Tca8113 cells were treated as described in (C). The mRNA levels of IL-1β were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (F) Quantitative data of (E). * P < 0.05 compared with the IL-1β-treated group. (G) The effect of ERK inhibition on long-term CXCR4 mRNA expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of ERK inhibitor U0126 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The mRNA levels of CXCR4 were measured by RT-PCR. β-actin mRNA levels were measured as loading controls. (I) The effect of ERK inhibition on CXCR4 protein expression induced by IL-1β. Tca8113 cells, pre-treated with the indicated concentrations of ERK inhibitor U0126 for 30 min, were treated with 20 ng/ml IL-1β for 24 h. The protein levels of CXCR4 were measured by western blot. β-actin protein levels were measured as loading controls.

Article Snippet: Recombinant human IL-1β, IL-1Ra, and mouse anti-human CXCR4 antibody (FACS) were purchased from R&D systems (Minneapolis, MN).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Inhibition, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Glycosphingolipids Promote Entry of a Broad Range of Human Immunodeficiency Virus Type 1 Isolates into Cell Lines Expressing CD4, CXCR4, and/or CCR5

doi:

Figure Lengend Snippet: Flow cytometry of CD4 and chemokine receptor expression in control and PPMP-treated cells. Expression levels of CD4, CXCR4, and CCR5 in GHOST-345 cells were examined by using control (left panel) and PPMP-treated (right panel) cells. Cells were incubated for 1 h at 4°C with PE-conjugated mouse IgG anti-CD4 (RPA-T4) (A), PE-conjugated mouse IgG anti-CXCR4 (12G5) (B), or PE-conjugated mouse IgG anti-CCR5 (2D7) (C). Fluorescence was examined with a Becton Dickinson FACScalibur at 10,000 events/sample. Control and PPMP-treated unlabeled cells were run as background. The surface concentrations of CD4, CXCR4, and CCR5 estimated from the observed median fluorescence intensities relative to median intensities for cells with known amounts of those receptors bound to their specific antibodies are about 5.2 × 104, 2.3 × 105, and 1.1 × 106 molecules/cell, respectively.

Article Snippet: Phycoerythrin (PE)-conjugated mouse immunoglobulin G (IgG) anti-CD4 (RPA-T4), PE-conjugated mouse IgG anti-CXCR4 (12G5), or PE-conjugated mouse IgG anti-CCR5 (2D7) from Pharmingen (San Diego, Calif.) was then added to each sample at a 1:5 dilution.

Techniques: Flow Cytometry, Expressing, Incubation, Fluorescence

Journal:

Article Title: Glycosphingolipids Promote Entry of a Broad Range of Human Immunodeficiency Virus Type 1 Isolates into Cell Lines Expressing CD4, CXCR4, and/or CCR5

doi:

Figure Lengend Snippet: Effect of GSL depletion on the CD4-chemokine receptor association. (A) CD4-CCR5 association. 3T3-CD4-CCR5 cells treated (+) and not treated (−) with PPMP were solubilized, and CD4 was isolated by coimmunoprecipitation by the anti-CCR5 antibody 5C7. The gels shown are Western blots obtained with rabbit anti-CD4 and goat anti-CCR5 antibody. The numbers below the gels represent intensity ratios determined using a Molecular Imager (Bio-Rad). (B) CD4-CXCR4-gp120 association. 3T3-CD4-CXCR4 cells treated (+) and not treated (−) with PPMP were solubilized, and CD4 was isolated by coimmunoprecipitation by anti-CXCR4 antibody 4G10 in the presence (+) or absence (−) of rgp120IIIB. The gels shown are Western blots obtained with rabbit anti-CD4 and mouse anti-CXCR4 antibody. The numbers below the gels represent intensity ratios determined using a Molecular Imager. (C) Inhibition of fusion with cells expressing the R5-utilizing envelope glycoprotein (Ba-L). (D) Inhibition of fusion with cells expressing the X4-utilizing envelope glycoprotein (IIIB).

Article Snippet: Phycoerythrin (PE)-conjugated mouse immunoglobulin G (IgG) anti-CD4 (RPA-T4), PE-conjugated mouse IgG anti-CXCR4 (12G5), or PE-conjugated mouse IgG anti-CCR5 (2D7) from Pharmingen (San Diego, Calif.) was then added to each sample at a 1:5 dilution.

Techniques: Isolation, Western Blot, Inhibition, Expressing

Journal:

Article Title: Deletion of the COOH-Terminal Domain of CXC Chemokine Receptor 4 Leads to the Down-regulation of Cell-to-Cell Contact, Enhanced Motility and Proliferation in Breast Carcinoma Cells

doi: 10.1158/0008-5472.CAN-05-3579

Figure Lengend Snippet: Overexpression of CXCR4-WT and CXCR4-ΔCTD in MCF-7 cells. A, map of the recombinant retroviral vector, pBMN-IRES-EGFP. PCR products encoding CXCR4-WT (1,071 bp) and CXCR4-ΔCTD (967 bp) were subcloned into the vector. LTR, long terminal repeat; Psi (ψ), consensus sequence for viral packaging. B, top, ethidium bromide–stained RNA after agarose gel electrophoresis. Total RNA was isolated from (1) MCF-7 cells, (2) vector-transduced MCF-7 cells (MCF-7/Vector), (3) MCF-7/CXCR4-WT cells, and (4) MCF-7/CXCR4-ΔCTD cells. 28S and 18S, rRNA; EB, ethidium bromide. Middle, PCR primer sets designed to amplify CXCR4 cDNA. A primer set for the 5′-terminus of CXCR4 was used to amplify a 465-bp CXCR4 cDNA fragment (21–485 nt), and a primer set for the 3′-terminus was used to amplify a 616-bp CXCR4 cDNA fragment (425–1,040 nt). Bottom, ethidium bromide–stained reverse transcription-PCR products. DNase-treated RNA (1 μg) was denatured at 70°C for 5 minutes and then reverse transcribed in 25 μL of a reaction mixture containing 1 μmol/L oligo(dT)16 primer, 5 units avian myeloblastosis virus (AMV) reverse transcriptase (AMV-RT) with AMV-RT buffer (Promega), and 0.2 mmol/L deoxynucleotide triphosphate. The synthesized cDNA (1 μL) was amplified by PCR using a primer set for GAPDH (5′-TCATTGACCTCAACTACATGG-3′ and 5′-GAGTCCTTCCACGATACCAAA-3′, PCR product: 413 bp, 110–522 nt in open reading frame from NM_002046), a primer set for the 5′-terminal sequence of CXCR4′ (5′-CACTTCAGATAACTACACCG-3′ and 5′-ATCCAGACGCCAACATAGAC-3′, PCR product: 465 bp, 21–485 nt in open reading frame from NM_003467), and a primer set for 3′-terminal sequence of CXCR4 (5′-CAACAGTCAGAGGCCAAGG-3′ and 5′-GAAGACTCAGACTCAGTGG-3′, PCR product: 616 bp, 425–1,040 nt). PCR reactions were done thrice. Representative gel.

Article Snippet: The primary antibodies used were αE-cadherin (610181), αZO-1 (610966), αCXCR4 (clone 12G5, MAB170, R&D Systems), αCXCR4 (clone 44708, MAB171, R&D Systems), anti-β-arrestin2 (sc-6387, Santa Cruz Biotechnology), and αRab11a (a gift from Dr. James Goldenring, Vanderbilt University Medical School, Nashville, TN).

Techniques: Over Expression, Recombinant, Retroviral, Plasmid Preparation, Sequencing, Staining, Agarose Gel Electrophoresis, Isolation, Reverse Transcription, Virus, Synthesized, Amplification

Journal:

Article Title: Deletion of the COOH-Terminal Domain of CXC Chemokine Receptor 4 Leads to the Down-regulation of Cell-to-Cell Contact, Enhanced Motility and Proliferation in Breast Carcinoma Cells

doi: 10.1158/0008-5472.CAN-05-3579

Figure Lengend Snippet: MCF-7/CXCR4-ΔCTD lost cell-to-cell contact. A, down-regulation of the ZO-1 in MCF-7/CXCR4-ΔCTD cells. MCF-7/Vector, MCF-7/CXCR4-WT, and MCF-7/CXCR4-ΔCTD cells were plated on glass coverslips and were fixed, permeabilized, and stained with an αZO-1 antibody followed by Alexa 594–conjugated mouse IgG, a secondary antibody. Representative Z-sectioned images (0.1 μm thick) from multiple individual experiments. B, down-regulation of E-cadherin in MCF-7/CXCR4-ΔCTD cells. MCF-7/Vector, MCF-7/CXCR4-WT, and MCF-7/CXCR4-ΔCTD cells were plated on glass coverslips and processed as stated above. Cells were stained with an αE-cadherin (E-cad) antibody followed by Alexa 594–conjugated α-mouse IgG. Representative Z-sectioned images (0.1 μm thick) from multiple individual experiments. C, protein lysates from MCF-7/Vector, MCF-7/CXCR4-WT, and MCF-7/CXCR4-ΔCTD cells were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was cut at around the 60-kDa marker into two pieces containing either high or low molecular weight proteins. The proteins on the membranes were subjected to immunoblot analysis with either an αE-cadherin or an αZO-1 antibody followed by an Alexa 680–conjugated α-mouse IgG for the high molecular weight proteins or an α-actin antibody followed by an Alexa 680–conjugated α-goat antibody for the low molecular weight proteins. Immunoreactive bands were visualized by scanning the emitted IR images using the Odyssey System.

Article Snippet: The primary antibodies used were αE-cadherin (610181), αZO-1 (610966), αCXCR4 (clone 12G5, MAB170, R&D Systems), αCXCR4 (clone 44708, MAB171, R&D Systems), anti-β-arrestin2 (sc-6387, Santa Cruz Biotechnology), and αRab11a (a gift from Dr. James Goldenring, Vanderbilt University Medical School, Nashville, TN).

Techniques: Plasmid Preparation, Staining, SDS Page, Membrane, Marker, Molecular Weight, Western Blot, High Molecular Weight

Journal:

Article Title: Deletion of the COOH-Terminal Domain of CXC Chemokine Receptor 4 Leads to the Down-regulation of Cell-to-Cell Contact, Enhanced Motility and Proliferation in Breast Carcinoma Cells

doi: 10.1158/0008-5472.CAN-05-3579

Figure Lengend Snippet: Subcellular distribution and trafficking of CXCR4-WT and CXCR4-DCTD. A, CXCR4-ΔCTD exhibited increased intracellular localization. MCF-7/Vector, MCF-7/CXCR4-WT, and MCF-7/CXCR4-ΔCTD cells were grown on glass coverslips in complete growth medium, fixed, permeabilized, and stained with an αCXCR4 antibody (clone 12G5, MAB170) followed by an Alexa 594–conjugated α-mouse IgG. Fluorescent images were captured with 0.1 μm Z-stack using a Zeiss Axiophot upright microscope. The expression of CXCR4 on cell surface was analyzed by FACS analysis using antibodies as indicated. B, truncated CXCR4 interacts and colocalizes with endogenous β-arrestin (β-arr). Cells were serum starved overnight and stimulated with/without 500 ng/mL CXCL12 for 5 minutes at 37°C. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. CXCR4 was probed with mouse monoclonal antibody (clone 12G5, MAB170) and β-arrestin2 with goat polyclonal anti-β-arrestin2 antibody (cross-reacts to a lesser extent to β-arrestin1, sc-6387). The receptor was visualized with Cy3 donkey anti-mouse antibody (715-165-150, Jackson ImmunoResearch) and β-arrestin2 was visualized through sequential binding of rabbit anti-goat antibody (BA-5000, Vector Laboratories, Burlingame, CA) and Cy5-donkey anti-rabbit antibody (711-175-152, Jackson ImmunoResearch). White arrows, vesicles where CXCR4 receptor and β-arrestin2 colocalize in transduced MCF-7 cells.

Article Snippet: The primary antibodies used were αE-cadherin (610181), αZO-1 (610966), αCXCR4 (clone 12G5, MAB170, R&D Systems), αCXCR4 (clone 44708, MAB171, R&D Systems), anti-β-arrestin2 (sc-6387, Santa Cruz Biotechnology), and αRab11a (a gift from Dr. James Goldenring, Vanderbilt University Medical School, Nashville, TN).

Techniques: Plasmid Preparation, Staining, Microscopy, Expressing, Binding Assay

Journal:

Article Title: Deletion of the COOH-Terminal Domain of CXC Chemokine Receptor 4 Leads to the Down-regulation of Cell-to-Cell Contact, Enhanced Motility and Proliferation in Breast Carcinoma Cells

doi: 10.1158/0008-5472.CAN-05-3579

Figure Lengend Snippet: MCF-7/CXCR4-ΔCTD cells exhibited increased cell motility. A, wound closure cell motility assay. MCF-7/Vector, MCF-7/CXCR4-WT, and MCF-7/CXCR4-ΔCTD cells were allowed to reach confluence in complete growth medium on glass coverslips in six-well plates and then scratched with a pipette tip to make wounds (a, c, and e). The closure of the wounds was monitored by microscopy after 18 hours (b, d, and f). Representative data from two individual experiments. B, quantitation of CXCL12 secreted into the medium. The endogenous secretions of CXCL12 (pg/105 cells) from MCF-7/Vector, MCF-7/CXCR4-WT, and MCF-7/CXCR4-ΔCTD cells in complete growth medium for 18 hours were measured by the ELISA analysis. The ELISA value in serum-free medium was not detected (data not shown). Representative data from two individual experiments. C, chemokinesis assay. Chemokinesis was measured using a 96-well chamber and 10 μm pore polycarbonate membrane filter. The filter membrane was soaked in a assay buffer solution (DMEM with 1 mg/mL BSA) containing 1 mg/mL collagen IV for 2 hours. MCF-7/Vector, MCF-7/CXCR4-WT, and MCF-7/CXCR4-ΔCTD cells were lifted with the Cell Dissociation Buffer, and 105 cells in 200 μL chemotaxis buffer were plated in the top chamber. Cells were incubated in the assay buffer with 50 ng/mL CXCL12 in both upper and lower chambers. After 4.5-hour incubation, cells migrated to the underside of the filters and were fixed with Diff-Quik (DADE Behring, Inc., Miami, FL). They were then stained with 1% crystal violet and counted by bright-field microscopy at ×200 magnification in five random fields. Representative data from one of three experiments. D, chemotaxis assay. The chemotaxis assay was done as the same condition/preparation as the chemokinesis assay as stated above, except that 0 to 250 ng/mL CXCL12 was added only in the lower chamber. Representative data from one of three experiments.

Article Snippet: The primary antibodies used were αE-cadherin (610181), αZO-1 (610966), αCXCR4 (clone 12G5, MAB170, R&D Systems), αCXCR4 (clone 44708, MAB171, R&D Systems), anti-β-arrestin2 (sc-6387, Santa Cruz Biotechnology), and αRab11a (a gift from Dr. James Goldenring, Vanderbilt University Medical School, Nashville, TN).

Techniques: Motility Assay, Plasmid Preparation, Transferring, Microscopy, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Membrane, Chemotaxis Assay, Incubation, Diff-Quik, Staining

Journal:

Article Title: Deletion of the COOH-Terminal Domain of CXC Chemokine Receptor 4 Leads to the Down-regulation of Cell-to-Cell Contact, Enhanced Motility and Proliferation in Breast Carcinoma Cells

doi: 10.1158/0008-5472.CAN-05-3579

Figure Lengend Snippet: MCF-7/CXCR4-ΔCTD cells exhibited increased cell proliferation. A, MTT assay for MCF-7/Vector, MCF-7/CXCR4-WT, and MCF-7/CXCR4-ΔCTD cells. Cells were plated in 24-well plates (2 × 104 per well) and incubated with MTT for 4 hours every 5 days. The product, formazan crystal, was dissolved in isopropanol containing 0.4 N hydrochloric acid, and the absorption at 570 nm was measured by a spectrophotometer. Each measure of absorbance was normalized by subtracting the nonspecific absorbance at 590 nm. SEs were derived using data from triplicate wells. The normalized absorbance value at day 0 was set as 1, and the following normalized measurements were represented as the fold increase. During cell culture, the culture medium was changed every 2 days. Representative data from three individual experiments. B and C, constitutive ERK2 activation in CXCR4-ΔCTD cells. Cells were treated with 50 μmol/L MEKK inhibitor PD98059 for 3 hours or 100 ng/mL CXCL12 (human SDF-1α) before cell lysis. The antibodies (200 ng/mL) used were αERK2 (sc-1647) and α-phosphorylated ERK1/2 (V-8031). Immunoreactive protein bands were visualized by scanning the emitted IR images of Alexa 680– and Alexa 800–conjugated secondary antibodies using the Odyssey System.

Article Snippet: The primary antibodies used were αE-cadherin (610181), αZO-1 (610966), αCXCR4 (clone 12G5, MAB170, R&D Systems), αCXCR4 (clone 44708, MAB171, R&D Systems), anti-β-arrestin2 (sc-6387, Santa Cruz Biotechnology), and αRab11a (a gift from Dr. James Goldenring, Vanderbilt University Medical School, Nashville, TN).

Techniques: MTT Assay, Plasmid Preparation, Incubation, Spectrophotometry, Derivative Assay, Cell Culture, Activation Assay, Lysis

Journal:

Article Title: Deletion of the COOH-Terminal Domain of CXC Chemokine Receptor 4 Leads to the Down-regulation of Cell-to-Cell Contact, Enhanced Motility and Proliferation in Breast Carcinoma Cells

doi: 10.1158/0008-5472.CAN-05-3579

Figure Lengend Snippet: Differential gene expression revealed that mesenchymal-related genes are up-regulated and epithelial-related genes are down-regulated in MCF-7/CXCR4-ΔCTD cells. A, RNA integrity index. Total RNA was isolated from MCF-7/Vector, MCF-7/CXCR4-WT, and MCF-7/ΔCTD cells and treated with DNase. The electrophoresis of RNA in the Eukaryote Total RNA Nano-DE114000902 resulted in a 99 percentile RNA integrity index value. B, list of differentially expressed genes identified by microarray analysis. MCF-7/CXCR4-WT-derived total RNA was reverse transcribed into cDNA with Cy5 labeling. MCF-7/Vector-derived RNA and MCF-7/CXCR4-ΔCDT-derived RNA were reverse transcribed into cDNA with Cy3 labeling. The labeled cDNA was hybridized to a human 30,000 oligoarray and the fluorescent ratio, Cy5/Cy3, was analyzed with GeneSpring software. The array data in Supplementary Data are represented as the fold change in gene expression comparing the gene expression in MCF-7/CXCR4-WT cells with that in MCF-7/Vector cells and comparing the gene expression in MCF-7/CXCR4-WT cells with that in MCF-7/CXCR4-ΔCTD cells. The genes possibly related to cell proliferation and EMT in this array analysis are listed in the table and the relationship between these genes is graphed (Fig. 5B). WT, MCF-7/CXCR4-WT cells; ΔCTD, MCF-7/CXCR4-ΔCTD cells; Vector, MCF-7/Vector cells; ↔, not a significant differential expression ratio (<3-fold) of Cy5/Cy3. In Supplementary Data I and II, all up-regulated or down-regulated genes with >3-fold differences in expression are listed.

Article Snippet: The primary antibodies used were αE-cadherin (610181), αZO-1 (610966), αCXCR4 (clone 12G5, MAB170, R&D Systems), αCXCR4 (clone 44708, MAB171, R&D Systems), anti-β-arrestin2 (sc-6387, Santa Cruz Biotechnology), and αRab11a (a gift from Dr. James Goldenring, Vanderbilt University Medical School, Nashville, TN).

Techniques: Gene Expression, Isolation, Plasmid Preparation, Electrophoresis, Microarray, Derivative Assay, Reverse Transcription, Labeling, Software, Quantitative Proteomics, Expressing