rabbit anti human cxcr6  (ProSci Incorporated)

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Novel extracellular role of REIC/Dkk-3 protein in PD-L1 regulation in cancer cells

doi: 10.1007/s00109-023-02292-w

Figure Lengend Snippet: Candidate-based screening of the REIC/Dkk-3 interaction. a HEK293T cells were co-transfected with the following Myc-tagged REIC/Dkk-3 and Flag-tagged chemokine receptors: EP1-3 (prostaglandin E receptor 1–3: gene name, PTGER1-3 ), LTR1-2 (cysteinyl leukotriene receptor 1, 2: gene names, CYSLTR1, 2 ), DP1, 2 (prostaglandin D2 receptor: gene names, PTGDR, PTGDR2 ), FP (prostaglandin F receptor: gene name, PTGFR ), IP (prostaglandin I2 receptor: gene name, PTGIR ), LPAR1-6 (lysophosphatidic acid receptor 1–6: gene name, LPAR1-6 ), C3aR (complement C3a receptor 1: gene name, C3AR1 ), C5aR (complement C5a receptor 1: gene name, C5AR ), FPR1 (formyl peptide receptor 1: gene name, FPR1 ), and CXCR1-6 (C-X-C motif chemokine receptor 1–6: gene name, CXCR1-6 ). After the immunoprecipitation of the expressed REIC/Dkk-3 with Myc antibody-conjugated beads, interacting receptors were detected by the Flag antibody. b Co-transfection of REIC/Dkk-3-3HA-6His with individual membrane proteins (C3aR, C5aR, CXCR2, CXCR6) tagged with 3Flag-6His or GFP was performed in HEK293T cells. 24 h after the transfections, cell extracts were prepared and then immunoprecipitated using anti-HA tag beads for the expressed REIC/Dkk-3, and a 4-μl volume of each specimen was analyzed by western blotting. c The binding of REIC/Dkk-3 to the identified REIC receptor candidates was expected to induce activation of some signal transduction pathways that lead to phosphorylation enhancements of several effector kinases. d HEK293T cells were transiently transfected with the identified candidate receptors for 24 h and then treated or not treated with REIC/Dkk-3 (10 ng/ml) for 1 h. The treated cells were lysed and subjected to western blotting with the indicated antibodies

Article Snippet: The antibodies used were as follows: mouse anti-HA tag antibody (clone 6E2; Cell Signaling Technology [CST], Danvers, MA), mouse anti-Myc tag antibody (clone 9B11; CST), mouse anti-Flag tag antibody (clone M2; Sigma-Aldrich), rabbit anti-human phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), rabbit anti-human p44/42 MAPK (Erk1/2), rabbit anti-human phospho-SAPK/JNK (Thr183/Tyr185), rabbit anti-human SAPK/JNK (CST), rabbit anti-human phospho-Akt (Ser473), rabbit anti-human Akt, mouse anti-human C5aR antibody (Bio-Rad Laboratories, Hercules, CA), rabbit anti-human CXCR2 antibody (Thermo Fisher Scientific), rabbit anti-human CXCR6 antibody (GeneTex, Irvine, CA), rabbit anti-human CXCR7 antibody (Proteintech, Rosemon, IL), mouse anti-human CMTM6 antibody (used as ab1) (Absea Biotechnology, China), rabbit anti-human CMTM6 antibody (used as ab2) (Sigma-Aldrich), mouse anti-GFP antibody (Thermo Fisher Scientific), rabbit anti-GAPDH (CST), mouse anti-α-tubulin (Sigma-Aldrich), and mouse anti-β-actin (Sigma-Aldrich) antibodies.

Techniques: Transfection, Immunoprecipitation, Cotransfection, Membrane, Western Blot, Binding Assay, Activation Assay, Transduction, Phospho-proteomics

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Novel extracellular role of REIC/Dkk-3 protein in PD-L1 regulation in cancer cells

doi: 10.1007/s00109-023-02292-w

Figure Lengend Snippet: Identification of protein(s) that interact with CXCR6 under the regulation of REIC/Dkk-3. a Individual cancer cell lines (BxPC-3, HeLa, and MDA-MB-231) were transfected with the expression plasmids (CXCR6-3Flag-6His and REIC/Dkk-3-3HA-6His) in the indicated combinations. After 36 h, these cells were lysed and pull-downed for the expressed foreign CXCR6, and the precipitated specimens were subjected to SDS-PAGE and then silver staining. b A band for a protein that bound with CXCR6 and whose binding was diminished by the presence of REIC/Dkk-3 was subjected to a liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis; the results are displayed as a modified list. c Co-transfection of PD-L1-3myc-6His with individual membrane proteins (PD-1, C3aR, C5aR, CXCR2, CXCR6) tagged with 3Flag-6His or GFP was performed in HEK293T cells. 24 h after the transfections, cell extracts were prepared and then immunoprecipitated using anti-myc tag beads for the expressed PD-L1, and a 4-μl volume of each specimen was analyzed by western blotting

Article Snippet: The antibodies used were as follows: mouse anti-HA tag antibody (clone 6E2; Cell Signaling Technology [CST], Danvers, MA), mouse anti-Myc tag antibody (clone 9B11; CST), mouse anti-Flag tag antibody (clone M2; Sigma-Aldrich), rabbit anti-human phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), rabbit anti-human p44/42 MAPK (Erk1/2), rabbit anti-human phospho-SAPK/JNK (Thr183/Tyr185), rabbit anti-human SAPK/JNK (CST), rabbit anti-human phospho-Akt (Ser473), rabbit anti-human Akt, mouse anti-human C5aR antibody (Bio-Rad Laboratories, Hercules, CA), rabbit anti-human CXCR2 antibody (Thermo Fisher Scientific), rabbit anti-human CXCR6 antibody (GeneTex, Irvine, CA), rabbit anti-human CXCR7 antibody (Proteintech, Rosemon, IL), mouse anti-human CMTM6 antibody (used as ab1) (Absea Biotechnology, China), rabbit anti-human CMTM6 antibody (used as ab2) (Sigma-Aldrich), mouse anti-GFP antibody (Thermo Fisher Scientific), rabbit anti-GAPDH (CST), mouse anti-α-tubulin (Sigma-Aldrich), and mouse anti-β-actin (Sigma-Aldrich) antibodies.

Techniques: Transfection, Expressing, SDS Page, Silver Staining, Binding Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Modification, Cotransfection, Membrane, Immunoprecipitation, Western Blot

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Novel extracellular role of REIC/Dkk-3 protein in PD-L1 regulation in cancer cells

doi: 10.1007/s00109-023-02292-w

Figure Lengend Snippet: The relationships among REIC/Dkk-3, the identified receptors (C5aR, CXCR2, and CXCR6), and PD-L1. a Co-transfection of REIC/Dkk-3-3HA-6His with individual membrane proteins (C3aR, C5aR, CXCR2, CXCR6, PD-L1, and PD1) tagged with 3Flag-6His was performed in HEK293T cells. 24 h after the transfections, cell extracts were prepared and then immunoprecipitated using anti-HA tag beads for the expressed REIC/Dkk-3, and a 4-μl volume of each specimen was analyzed by western blotting. b, c HEK293T cells were transfected with the indicated plasmids in different combinations. Cell pellets were collected after 24 h, lysed, immunoprecipitated using anti-Myc tag beads for the expressed PD-L1, and analyzed by western blotting with ( b ) and without ( c ) the immunoprecipitation. For the immunoprecipitated samples ( b ), the sample volume from the REIC-Dkk-3 co-transfection group was 20 μl, which matched a fivefold sample volume from the control GFP co-transfection group. Western blotting was performed using the same volume from the same concentration of the samples adjusted through all preparations ( c ). d Schematic representation of the relation among REIC/Dkk-3, REIC receptors, and PD-L1. REIC/Dkk-3 was expected to exert PD-L1 degradation through the binding with the REIC/Dkk-3-target receptors

Article Snippet: The antibodies used were as follows: mouse anti-HA tag antibody (clone 6E2; Cell Signaling Technology [CST], Danvers, MA), mouse anti-Myc tag antibody (clone 9B11; CST), mouse anti-Flag tag antibody (clone M2; Sigma-Aldrich), rabbit anti-human phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), rabbit anti-human p44/42 MAPK (Erk1/2), rabbit anti-human phospho-SAPK/JNK (Thr183/Tyr185), rabbit anti-human SAPK/JNK (CST), rabbit anti-human phospho-Akt (Ser473), rabbit anti-human Akt, mouse anti-human C5aR antibody (Bio-Rad Laboratories, Hercules, CA), rabbit anti-human CXCR2 antibody (Thermo Fisher Scientific), rabbit anti-human CXCR6 antibody (GeneTex, Irvine, CA), rabbit anti-human CXCR7 antibody (Proteintech, Rosemon, IL), mouse anti-human CMTM6 antibody (used as ab1) (Absea Biotechnology, China), rabbit anti-human CMTM6 antibody (used as ab2) (Sigma-Aldrich), mouse anti-GFP antibody (Thermo Fisher Scientific), rabbit anti-GAPDH (CST), mouse anti-α-tubulin (Sigma-Aldrich), and mouse anti-β-actin (Sigma-Aldrich) antibodies.

Techniques: Cotransfection, Membrane, Transfection, Immunoprecipitation, Western Blot, Control, Concentration Assay, Binding Assay

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Novel extracellular role of REIC/Dkk-3 protein in PD-L1 regulation in cancer cells

doi: 10.1007/s00109-023-02292-w

Figure Lengend Snippet: Expression profile of the molecules of interest. a, b Total RNAs ( a ) and protein extracts ( b ) prepared from the indicated cell lines (non-cancerous human HEK293T embryonic kidney epithelial cells; human pancreatic cancer PANC-1, AsPC-1, and MIA PaCa-2 cells; human breast cancer MDA-MB-231 and MCF-7 cells; human prostate cancer PC-3 and LNCaP cells; human squamous cancer A431 cells; human cervical cancer HeLa cells; human melanoma MeWo cells; human mesothelioma MSTO-211H cells; human lung cancer NCI-H2170 cells; and human isolated neutrophils from a healthy donor) were analyzed for the expressions of REIC/Dkk-3 and PD-L1. c, d The selected PD-L1 highly positive cell lines (MIA PaCa-2 cells; MDA-MB-231cells; LNCaP cells; A431 cells; HeLa cells; MeWo cells; MSTO-211H cells) were further analyzed for their expressions of C5aR, CXCR2, CXCR6, CXCR7, and CMTM6 in comparison to those in HEK293T cells and human isolated neutrophils from a healthy donor by quantitative real-time PCR ( c ) and western blotting ( d ). ACTB mRNA was used as a control for the analysis ( c ). Data are mean ± SD. ND, not detected; * p < 0.05, ** p < 0.01 by Student’s t- test

Article Snippet: The antibodies used were as follows: mouse anti-HA tag antibody (clone 6E2; Cell Signaling Technology [CST], Danvers, MA), mouse anti-Myc tag antibody (clone 9B11; CST), mouse anti-Flag tag antibody (clone M2; Sigma-Aldrich), rabbit anti-human phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), rabbit anti-human p44/42 MAPK (Erk1/2), rabbit anti-human phospho-SAPK/JNK (Thr183/Tyr185), rabbit anti-human SAPK/JNK (CST), rabbit anti-human phospho-Akt (Ser473), rabbit anti-human Akt, mouse anti-human C5aR antibody (Bio-Rad Laboratories, Hercules, CA), rabbit anti-human CXCR2 antibody (Thermo Fisher Scientific), rabbit anti-human CXCR6 antibody (GeneTex, Irvine, CA), rabbit anti-human CXCR7 antibody (Proteintech, Rosemon, IL), mouse anti-human CMTM6 antibody (used as ab1) (Absea Biotechnology, China), rabbit anti-human CMTM6 antibody (used as ab2) (Sigma-Aldrich), mouse anti-GFP antibody (Thermo Fisher Scientific), rabbit anti-GAPDH (CST), mouse anti-α-tubulin (Sigma-Aldrich), and mouse anti-β-actin (Sigma-Aldrich) antibodies.

Techniques: Expressing, Isolation, Comparison, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: HCT-116 colorectal cancer cells secrete chemokines which induce chemoattraction and intracellular calcium mobilization in NK92 cells

doi: 10.1007/s00262-019-02319-7

Figure Lengend Snippet: Expression of CCR10 and CXCR6 levels using flow cytometry, western blot, and qRT-PCR. NK92 cells were either pretreated with DMSO, or with 100 µM of MMF or DMF. a NK92 cells were washed, and the expression of CCR10 or CXCR6 or CCR3 was examined using flow cytometry. Percentages of positive cells are shown on the y-axis and staining with isotype control antibodies is shown in blue. One of two representative experiments is shown. b Immunoblot analysis showing the protein levels of CCR10 and CXCR6 as compared to the level of the house-keeping β-actin. c qRT-PCR analysis showing the receptors’ mRNA expression in NK92 cells after treatment with MMF or DMF as normalized to the house-keeping 18S

Article Snippet: Mouse anti-human CCR10 was purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA), rabbit anti-human CXCR6 was from Abcam (Cambridge, UK) and mouse anti-human β-actin from Sigma-Aldrich.

Techniques: Expressing, Flow Cytometry, Western Blot, Quantitative RT-PCR, Staining

Journal: Oncology Letters

Article Title: Expression analysis and clinical significance of CXCL16/CXCR6 in patients with bladder cancer

doi: 10.3892/ol.2012.976

Figure Lengend Snippet: Table I

Article Snippet: Following treatment with protein-blocking solution containing 10% bovine serum albumin (BioShop, Burlington, ON, Canada) in PBS to block non-specific binding, the sections were incubated for 1 h at room with rabbit anti-human CXCR6 (1 mg/ml, GeneTex Corporation, Zeeland, MI, USA) or rabbit anti-human CXCL16 (0.3 μ g/ml, PeproTech, Rocky Hill, NJ, USA) antibodies.

Techniques: Staining

Journal: international Journal of Oncology

Article Title: Expression and potential function of the CXC chemokine CXCL16 in pancreatic ductal adenocarcinoma

doi: 10.3892/ijo_00000009

Figure Lengend Snippet: Figure 1. Expression of CXCL16 and CXCR6 mRNA in pancreatic cancer (PDAC) cells and pancreatic tissues (qRT-PCR). (A) CXCL16 and (B) CXCR6 mRNA expression levels in pancreatic cancer cell lines. (C) CXCL16 mRNA expression levels in normal pancreas (n=10), chronic pancreatitis (CP) (n=11), and PDAC tissue (n=11); (P<0.001 PDAC versus normal, P<0.001 PDAC versus CP). (D) CXCR6 mRNA expression levels in normal pancreas (n=18), CP (n=32), and PDAC tissue (n=33); (P<0.001 PDAC versus normal, P<0.001 PDAC versus CP).

Article Snippet: Tissue or cellular protein lysates (25 μg) and 75 μg of spleen tissue serving as a positive control, were separated by electrophoresis in 10-12% Bis-Tris gels (Invitrogen), transferred to nitrocellulose membrane and blocked with 5% non-fat dry milk in TTBS (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween-20; 5% M-TTBS) for 1 h. The membranes were incubated overnight at 4 ̊C with goat anti-human CXCL16 (R&D Systems, Wiesbaden, Germany) or rabbit anti-human CXCR6 (ProSci, Poway, CA, USA), diluted 1:500 in 5% M-TTBS.

Techniques: Expressing, Quantitative RT-PCR

Journal: international Journal of Oncology

Article Title: Expression and potential function of the CXC chemokine CXCL16 in pancreatic ductal adenocarcinoma

doi: 10.3892/ijo_00000009

Figure Lengend Snippet: Figure 2. Expression of CXCL16 and CXCR6 protein in pancreatic cancer (PDAC) cells and pancreatic tissues. (A) CXCL16 and CXCR6 protein expression in pancreatic cancer cell lines. All tested cell lines displayed protein expression for CXCL16 (upper panel) and CXCR6 (lower panel). Spleen lysate served as a positive control. (B) CXCL16 protein expression in normal pancreas (n=4) and chronic pancreatitis (CP) (n=4) (lower panel), and pancreatic cancer (PDAC) tissue (n=8) (upper panel). Spleen lysate served as a positive control. In line with the results of the qRT-PCR, there is upregulated expression of CXCL16 protein in CP as well as in PDAC compared to normal pancreas. CXCR6 was not detectable in human tissue samples at the protein level using immunoblotting.

Article Snippet: Tissue or cellular protein lysates (25 μg) and 75 μg of spleen tissue serving as a positive control, were separated by electrophoresis in 10-12% Bis-Tris gels (Invitrogen), transferred to nitrocellulose membrane and blocked with 5% non-fat dry milk in TTBS (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween-20; 5% M-TTBS) for 1 h. The membranes were incubated overnight at 4 ̊C with goat anti-human CXCL16 (R&D Systems, Wiesbaden, Germany) or rabbit anti-human CXCR6 (ProSci, Poway, CA, USA), diluted 1:500 in 5% M-TTBS.

Techniques: Expressing, Positive Control, Quantitative RT-PCR, Western Blot

Journal: international Journal of Oncology

Article Title: Expression and potential function of the CXC chemokine CXCL16 in pancreatic ductal adenocarcinoma

doi: 10.3892/ijo_00000009

Figure Lengend Snippet: Figure 3. Expression and localization of CXCL16 and CXCR6 in pancreatic tissues. Immunohistochemistry was performed as described in Materials and methods. CXCL16 immunoreaction in normal pancreas (A), chronic pancreatitis (CP) (B), and pancreatic cancer (PDAC) (C). CXCR6 immunoreaction in normal pancreas (D), CP (E), PDAC (F). Note the negative immunoreactivity in consecutive negative control tissue sections (inserts). The given slides reveal representative examples of the immunohistochemistry of CXCL16 and CXCR6.

Article Snippet: Tissue or cellular protein lysates (25 μg) and 75 μg of spleen tissue serving as a positive control, were separated by electrophoresis in 10-12% Bis-Tris gels (Invitrogen), transferred to nitrocellulose membrane and blocked with 5% non-fat dry milk in TTBS (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween-20; 5% M-TTBS) for 1 h. The membranes were incubated overnight at 4 ̊C with goat anti-human CXCL16 (R&D Systems, Wiesbaden, Germany) or rabbit anti-human CXCR6 (ProSci, Poway, CA, USA), diluted 1:500 in 5% M-TTBS.

Techniques: Expressing, Immunohistochemistry, Negative Control