ccr1 antibody  (Bio-Techne corporation)

Journal: International Journal of Molecular Sciences

Article Title: Cytokine CCL9 Mediates Oncogenic KRAS-Induced Pancreatic Acinar-to-Ductal Metaplasia by Promoting Reactive Oxygen Species and Metalloproteinases

doi: 10.3390/ijms25094726

Figure Lengend Snippet: Elevated expression of CCL9 and its receptors in the ADM regions of pancreas tissue samples. ( A ) Pancreas tissues of p48 cre :Kras G12D mice and their matching littermate p48 cre mice were immunostained with antibodies of CCL9. Scale bar: 50 µm. Arrow: CCL9-positive immune cells. ( B ) The ADM area of p48 cre :Kras G12D mouse pancreas was fluorescently immunostained with antibodies of F4/80 (green, macrophage marker), CK-19 (grey, ductal marker) and CCL9 (Red). The cells were visualized by DAPI labeling (blue). Asterisk indicates the duct-like structures derived from acini (where ADM occurred). Scale bar: 25 µm. ( C ) Exactly that same as ( B ), except using the pancreas tissue of p48 cre mouse. ( D ) Pancreas tissues of p48 cre :Kras G12D mice and their matching littermate p48 cre mice were immunostained with antibodies of CCR1 and CCR3. Scale bar: 50 µm. ( E , F ) Human pancreas tissue samples that contain ADM areas and normal pancreas tissues were immunostained with antibodies of CCL15 ( E ), CCR1 and CCR3 ( F ). Scale bar: 50 µm.

Article Snippet: CCR1 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Expressing, Marker, Labeling, Derivative Assay

Journal: Oncotarget

Article Title: C-C motif chemokine receptor 1 (CCR1) is a target of the EGF-AKT-mTOR-STAT3 signaling axis in breast cancer cells

doi: 10.18632/oncotarget.21813

Figure Lengend Snippet: (A) Representative immunohistochemical staining of CCR1 in breast invasive ductal carcinoma (IDC) and adjacent normal tissue samples. Square N indicates total tissue number. (B) Relative staining scores of CCR1 immunoreactivity in IDC and adjacent normal tissues. Data are mean ± SD. Spot, individual sample; blue horizontal line, mean; red vertical line, error bar. **** , P < 0.0001 (n = 178, paired two-tailed t -test). (C) Relative immunohistochemical staining intensity of CCR1 in ER, PR, HER2, or TN negative (-) or positive (+) samples. Square number indicates total sample number. ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2; TN, triple negative (ER - /PR - /HER2 - ). Values are expressed as mean ± SD. NS , not significant; **** , P < 0.0001; ** , P < 0.0220 by one-way ANOVA followed by Sidak’s multiple comparisons test (comparison between normal and cancer groups).

Article Snippet: Immunohistochemical staining was performed with a Bond-Max autostainer system (Leica Microsystems, Bannockburn, IL, USA) using primary antibodies against CCR1 (1:150; Novus Biologicals, Littleton, CO, USA).

Techniques: Immunohistochemical staining, Staining, Two Tailed Test, Comparison

Journal: Oncotarget

Article Title: C-C motif chemokine receptor 1 (CCR1) is a target of the EGF-AKT-mTOR-STAT3 signaling axis in breast cancer cells

doi: 10.18632/oncotarget.21813

Figure Lengend Snippet: (A) CCR1 expression in various breast cancer cells. Steady-state CCR1 mRNA levels were examined by RT-PCR. GAPDH was used as an internal control. (B) Silencing of CCR1 by expression of lentiviral CCR1 shRNA (shCCR1). Knockdown of CCR1 expression was verified by RT-PCR. (C) Invasion assay. MDA-MB-231 transfectants expressing shCT or shCCR1 were cultured in three-dimensional spheroids in an extracellular matrix. Protrusion of invasive cells was captured with an Eclipse TS100 microscope equipped with a digital sight camera. (D) Experimental in vivo lung metastasis. Representative BLI imaging of nude mice bearing MDA-MB-231 (shCT-Luc and shCCR1-Luc) tumors with metastatic lung lesions. (E) Eight weeks after inoculation, BLI signal intensity was measured in MDA-MB-231 transfectants with Spectrum IVIS.

Article Snippet: Immunohistochemical staining was performed with a Bond-Max autostainer system (Leica Microsystems, Bannockburn, IL, USA) using primary antibodies against CCR1 (1:150; Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, shRNA, Invasion Assay, Cell Culture, Microscopy, In Vivo, Imaging

Journal: Oncotarget

Article Title: C-C motif chemokine receptor 1 (CCR1) is a target of the EGF-AKT-mTOR-STAT3 signaling axis in breast cancer cells

doi: 10.18632/oncotarget.21813

Figure Lengend Snippet: (A) MDA-MB-231 cells were serum-depleted overnight and treated with EGF (50 ng/mL) for 0–24 h, and total RNA was extracted. CCR1 mRNA levels were examined by RT-PCR. GAPDH was used as an internal control. (B) Serum-starved MDA-MB-231 cells were treated as in A, and CCR1 mRNA levels were assessed by quantitative real-time PCR ( Q-PCR ). Values were normalized to GAPDH mRNA levels. Data are mean ± SD ( n = 3). * , P = 0.0247; *** , P < 0.0009; **** , P < 0.0001 (compared to untreated control; by Sidak’s multiple comparisons test). (C) Serum-starved MDA-MB-231 cells were treated as in A, and CCR1 protein levels were assessed by immunoblotting. GAPDH was used as an internal control. (D) Flow cytometry. After serum-starved MDA-MB-231 cells were treated with EGF (50 ng/mL) for 12 h, CCR1 protein expression on the cell surface was measured using flow cytometry. (E) MDA-MB-231 cells were treated with EGF (50 ng/mL) for 12 h, and then incubated with an antibody against CCR1, and a AlexaFluor 555-conjugated ( red signal ) secondary antibody for 30 min. Nuclear DNA was stained with 0.1 μg/mL Hoechst 33258 for 10 min ( blue signal ). Fluorescence-positive cells were examined under an EVOSf1 ® fluorescence microscope.

Article Snippet: Immunohistochemical staining was performed with a Bond-Max autostainer system (Leica Microsystems, Bannockburn, IL, USA) using primary antibodies against CCR1 (1:150; Novus Biologicals, Littleton, CO, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Expressing, Incubation, Staining, Fluorescence, Microscopy

Journal: Oncotarget

Article Title: C-C motif chemokine receptor 1 (CCR1) is a target of the EGF-AKT-mTOR-STAT3 signaling axis in breast cancer cells

doi: 10.18632/oncotarget.21813

Figure Lengend Snippet: (A) MDA-MB-231 cells were transfected with 0.2 μg of the promoter-reporter plasmids along with 50 ng of the Renilla luciferase expression plasmid, pRL-null, for normalization of transfection efficiency. After 48 h, the cells were either treated with EGF (20 ng/mL) for 12 h or untreated (control), after which dual luciferase activity was measured. Data are means ± SD ( n = 3). (B) The nucleotide sequence of the promoter region (−200 to +27) of human CCR1 . (C) MDA-MB-231 cells were transfected with either wild-type pCCR1-Luc(−200/+27) or a STAT site mutant construct (mtSTAT), and with either an empty vector or a STAT3 expression plasmid. After 48 h, the cells were collected, and dual luciferase activity was measured. Data are means ± SD ( n = 3). **** , p < 0.0001. (D) MDA-MB-231 cells were transfected with either wild-type pCCR1-Luc(−200/+27) or mtSTAT. After 48 h, the cells were either treated with EGF (20 ng/mL) for 12 h or untreated, and then, dual luciferase activity was measured. Data are means ± SD ( n = 3). **** , p < 0.0001 by Sidak’s multiple comparisons test. (E) EMSA. MDA-MB-231 cells were treated with EGF (50 ng/mL) for 12 h or untreated, and nuclear extracts were prepared. EMSA was performed with biotin-labeled oligonucleotide probes containing the STAT3 binding sequence (5′-gttaacttggcttccaggaagtggc-3′) in the absence or presence of a 10-fold (10 × ) or 50-fold (50 × ) molar excess of unlabeled competitor oligonucleotides ( Comp ).

Article Snippet: Immunohistochemical staining was performed with a Bond-Max autostainer system (Leica Microsystems, Bannockburn, IL, USA) using primary antibodies against CCR1 (1:150; Novus Biologicals, Littleton, CO, USA).

Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Sequencing, Mutagenesis, Construct, Labeling, Binding Assay

Journal: Oncotarget

Article Title: C-C motif chemokine receptor 1 (CCR1) is a target of the EGF-AKT-mTOR-STAT3 signaling axis in breast cancer cells

doi: 10.18632/oncotarget.21813

Figure Lengend Snippet: (A) Serum-starved MDA-MB-231 cells were treated with EGF (50 ng/mL) in the absence or presence of different concentrations of Stattic. After 12 h, total RNA was extracted, and CCR1 mRNA levels were examined by RT-PCR. (B) Serum-starved MDA-MB-231 cells were treated as in A, and CCR1 mRNA levels were assessed by quantitative real-time PCR. Values were normalized to GAPDH mRNA levels. Data are means ± SD ( n = 3). **** , p < 0.0001 by Sidak’s multiple comparisons test. (C) Serum-starved MDA-MB-231 transfectants expressing control scrambled shRNA (shCT) or STAT3 shRNA (shSTAT3) were treated with EGF (50 ng/mL) for 12 h, and total RNA was extracted. CCR1 mRNA levels were examined by RT-PCR. GAPDH was used as an internal control. (D) Cells were treated as in C, and CCR1 mRNA levels were determined by quantitative real-time PCR. Data are means ± SD ( n = 3). * , p = 0.0157; **** , p < 0.0001 by Sidak’s multiple comparisons test.

Article Snippet: Immunohistochemical staining was performed with a Bond-Max autostainer system (Leica Microsystems, Bannockburn, IL, USA) using primary antibodies against CCR1 (1:150; Novus Biologicals, Littleton, CO, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, shRNA

Journal: Oncotarget

Article Title: C-C motif chemokine receptor 1 (CCR1) is a target of the EGF-AKT-mTOR-STAT3 signaling axis in breast cancer cells

doi: 10.18632/oncotarget.21813

Figure Lengend Snippet: (A) Serum-starved MDA-MB-231 cells were treated with EGF (50 ng/mL) for different times, and phosphorylation of STAT3 (Tyr705), STAT3 (Ser727), AKT (Ser473), or mTOR (Ser2481) was analyzed by immunoblotting. GAPDH was used as an internal control. (B) Serum-starved MDA-MB-231 cells were either pretreated with API2 (10, 20, or 50 μM) for 30 min or untreated, and then treated with EGF (50 ng/mL). After 15 min, the cells were collected, and protein lysates were prepared. Phosphorylation of AKT (Ser473), mTOR (Ser2481), STAT3 (Ser727), or ERK1/2 (Thr201/Tyr204) was analyzed by immunoblotting. GAPDH was used as an internal control. (C) Cells were treated as in (B). After 24 h, total RNAs were extracted, and CCR1 mRNA levels were examined by RT-PCR. GAPDH was used as an internal control. (D) Serum-starved MDA-MB-231 cells were treated as in B, and CCR1 mRNA levels were assessed by quantitative real-time PCR ( Q-PCR ). Values were normalized to GAPDH mRNA levels. Data are means ± SD ( n = 3). ** , p = 0.0027; **** , p < 0.0001 by Sidak’s multiple comparisons test.

Article Snippet: Immunohistochemical staining was performed with a Bond-Max autostainer system (Leica Microsystems, Bannockburn, IL, USA) using primary antibodies against CCR1 (1:150; Novus Biologicals, Littleton, CO, USA).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Oncotarget

Article Title: C-C motif chemokine receptor 1 (CCR1) is a target of the EGF-AKT-mTOR-STAT3 signaling axis in breast cancer cells

doi: 10.18632/oncotarget.21813

Figure Lengend Snippet: (A) AKT1 or AKT2 expression was examined in MDA-MB-231 cells expressing control shRNA (shCT), AKT1 shRNA (shAkt1), AKT2 shRNA (shAkt2), or AKT1 plus AKT2 [shAkt(1+2)] shRNAs. GAPDH was used as an internal control. (B) CCR1 mRNA expression was detected by RT-PCR in shCT, shAkt1, shAkt2, or shAkt(1+2)-expressing cells. GAPDH was used as an internal control. (C) CCR1 mRNA expression was detected by Q-PCR in shCT, shAkt1, shAkt2, or shAkt(1+2)-expressing cells. Values were normalized to GAPDH mRNA levels. Data are means ± SD ( n = 3). **** , p < 0.0001 by Sidak’s multiple comparisons test. (D) Serum-starved shCT or shAkt1 cells were either untreated or treated with EGF (50 ng/mL) for 15 or 30 min, and the phosphorylation status of mTOR (Ser2481) and STAT3 (Ser727) was analyzed by immunoblotting. GAPDH were used as internal controls. (E) Serum-starved shCT or shAkt1 cells were treated with EGF (50 ng/mL) for 24 h, and CCR1 mRNA expression was detected by RT-PCR. GAPDH was used as an internal control.

Article Snippet: Immunohistochemical staining was performed with a Bond-Max autostainer system (Leica Microsystems, Bannockburn, IL, USA) using primary antibodies against CCR1 (1:150; Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Oncotarget

Article Title: C-C motif chemokine receptor 1 (CCR1) is a target of the EGF-AKT-mTOR-STAT3 signaling axis in breast cancer cells

doi: 10.18632/oncotarget.21813

Figure Lengend Snippet: (A) Serum-starved MDA-MB-231 cells were either left untreated or pretreated with rapamycin (20 or 50 nM) for 30 min, and then treated with EGF (50 ng/mL). After 15 min, the cells were collected, and protein lysates were prepared. Lysates were immunoblotted with phospho-mTOR (Ser2481) and phospho-STAT3 (Ser727) antibodies. GAPDH was used as an internal control. (B) Serum-starved MDA-MB-231 cells were treated as in A. After 24 h, total RNAs were extracted, and CCR1 mRNA levels were examined by RT-PCR. GAPDH was used as an internal control. (C) Serum-starved MDA-MB-231 cells were treated as in B, and CCR1 mRNA levels were assessed by quantitative real-time PCR ( Q-PCR ). Values were normalized to GAPDH mRNA levels. Data are means ± SD ( n = 3). * , p = 0.0343; *** , p = 0.0004; **** , p < 0.0001 (compared with EGF-treated cells; by Sidak’s multiple comparisons test). (D) Silencing of mTOR by expression of lentiviral mTOR shRNA (shTOR). Knockdown of mTOR expression was verified by immunoblotting. (E) Serum-starved shCT and shTOR clones #2 and #5 cells were treated with EGF (50 ng/mL) for 24 h, and CCR1 mRNA expression was detected by RT-PCR. GAPDH was used as an internal control. (F) Serum-starved shCT and shTOR clones #2 and #5 cells were treated as in E, and CCR1 mRNA levels were assessed by Q-PCR. Values were normalized to GAPDH mRNA levels. Data are means ± SD ( n = 3). **** , p < 0.0001; NS , statistically not significant (by Sidak’s multiple comparisons test).

Article Snippet: Immunohistochemical staining was performed with a Bond-Max autostainer system (Leica Microsystems, Bannockburn, IL, USA) using primary antibodies against CCR1 (1:150; Novus Biologicals, Littleton, CO, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, shRNA, Western Blot, Clone Assay