antibodies against caix  (Novus Biologicals)

Journal: International Journal of Molecular Sciences

Article Title: PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity

doi: 10.3390/ijms21228545

Figure Lengend Snippet: C -terminal amino acid Ala459 residue cooperates in the regulation of CAIX catalytic function: ( a ) Immunoblot analysis of cell lysates from C33a cells transfected with mock control, CAIX wild type (wt) or mutant (A459G) cultured 48 h in hypoxia. CAIX was detected using mouse monoclonal antibody M75 diluted 1:10 and β-actin was detected using mouse monoclonal antibody (CS3700, Cell Signaling Technology, Danvers, Massachusetts) diluted 1:5000. HRP-conjugated anti-mouse antibody (Dako Agilent, Santa Clara, California) diluted 1:5000 was used as a secondary antibody. ( b ) Fluorescence staining of CAIX in non-fixed and non-permeabilized C33a-CAIX wild type as well as A459G transfectants measured by flow cytometry. CAIX was detected using PG-domain specific mouse monoclonal antibody M75 diluted to 1 µg/mL and AlexaFluor 488-conjugated anti-mouse secondary antibody (Invitrogen, Carlsbad, California) diluted 1:1000. Results clearly demonstrate plasma membrane localization of CAIX and showed that 42.7% of C33a-CAIX-wt and 50.9% of C33a-CAIX-A459G transfectants expressed CAIX protein. C33a cells transfected with mock control plasmid were used as a negative control. The data are presented as the mean, n = 2. ( c ) Effect of Ala459 mutation on CAIX-mediated extracellular acidification. The graph shows the differences between pHe values (ΔpH) of culture media from CAIX wt or A459G-transfected and mock-transfected cells cultured 48 h in hypoxia. A459G mutant reduced acidification of extracellular pHe when compared to control wild type C33a-CAIX transfectants. The data are presented as the mean ± s.d., n = 5. Statistical significance was analyzed using the Student’s t -test and expressed as a p -value (* p < 0.05). ( d ) Effect of Ala459 mutation on migration capacity of C33a cells. The graph depicts the results of the wound healing assay given as a % of the area covered by cells migrating to close the wound at 30 h after the scratch, measured at various positions along the wounds. Area covered by C33a cells expressing CAIX-wt was set as 100%. C33a cells expressing CAIX with mutated Ala459 exhibited slower migration. The data are presented as the mean ± s.d., n = 10. Statistical significance was analyzed using the Student’s t -test and expressed as a p -value (* p < 0.05). ( e ) Accumulation of the fluorescent sulfonamide (FITC-CA-i) occurred in hypoxic MDCK cells expressing the CAIX-wt, whereas it was diminished in hypoxic MDCK cell transfected with the CAIX-A459G mutant. Images were taken using objective 10×. ( f ) In situ detection of the interaction between CAIX and AE2 using a proximity ligation assay (PLA). Analysis was performed in C33a cells transiently transfected with CAIX-wt and A459G mutant and cultured in hypoxia for an additional 48 h. Red PLA signal indicates the existence of interaction or close proximity localization also between CAIX with mutated Ala459 and AE2. C33a-mock transfectants served as a negative assay control due to a lack of one target protein. CAIX protein was post-labelled in green, nuclei are blue. Images were taken using objective 40× and zoom 3.

Article Snippet: Samples were blocked with 3% BSA/PBS for 30 min, incubated with a mixture of antibodies against CAIX (NB100-417, Novus Biological, Centennial, Colorado; diluted to 1 µg/mL) and PIMT (sc-100977, Santa Cruz Biotechnology, Dallas, TX, USA; diluted to 2 µg/mL) or CAIX (M75, diluted to 1 µg/mL) and AE2 (GenScript, Piscataway, NJ, USA; diluted to 1 µg/mL) for 1 h, incubated with plus and minus PLA probes for 1 h, then incubated with a ligation mixture containing connector oligonucleotides for 30 min, and finally with an amplification mixture containing a fluorescently labelled DNA probe for 100 min.

Techniques: Residue, Western Blot, Transfection, Mutagenesis, Cell Culture, Fluorescence, Staining, Flow Cytometry, Membrane, Plasmid Preparation, Negative Control, Migration, Wound Healing Assay, Expressing, In Situ, Proximity Ligation Assay

Journal: International Journal of Molecular Sciences

Article Title: PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity

doi: 10.3390/ijms21228545

Figure Lengend Snippet: Identification of novel intracellular binding partner(s) of carbonic anhydrase IX: ( a ) Schematic outline of the experimental procedure. ( b ) Comparative analysis of proteomic data based on ProteinCenterTM analyzer (Thermo Fischer Scientific, Waltham, Massachusetts). Graphical scheme represents summary characterization and clustering of predicted binding partners selected from the comparison of wild type versus A459G sample data sets. Results proved a cluster of cytoplasmic proteins binding exclusively the wild type form of intracellular tail of CAIX (non-mutated Ala) with different molecular functions and roles in cellular processes. Among them, PIMT was characterized as a protein exhibiting catalytic activity with a function in metabolic processes.

Article Snippet: Samples were blocked with 3% BSA/PBS for 30 min, incubated with a mixture of antibodies against CAIX (NB100-417, Novus Biological, Centennial, Colorado; diluted to 1 µg/mL) and PIMT (sc-100977, Santa Cruz Biotechnology, Dallas, TX, USA; diluted to 2 µg/mL) or CAIX (M75, diluted to 1 µg/mL) and AE2 (GenScript, Piscataway, NJ, USA; diluted to 1 µg/mL) for 1 h, incubated with plus and minus PLA probes for 1 h, then incubated with a ligation mixture containing connector oligonucleotides for 30 min, and finally with an amplification mixture containing a fluorescently labelled DNA probe for 100 min.

Techniques: Binding Assay, Comparison, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity

doi: 10.3390/ijms21228545

Figure Lengend Snippet: Protein L-isoaspartyl methyltransferase interacts with carbonic anhydrase IX: ( a ) Coomassie staining of purified GST and GST-CAIX-IC-wt separated by SDS-PAGE (left panel). Immunoblot analysis of GST-CAIX-IC-wt immunoprecipitates and lysates from different cancer cell lines: C33a, Panc-1, A549, RKO and MDA-MB-231 (right panel). GST immunoprecipitates and beads after lysate preclearing was used as negative control. PIMT signal was detected using monoclonal antibody (sc-100977, Santa Cruz Biotechnology, Dallas, Texas) diluted 1:1000. ( b ) In situ detection of the interaction between PIMT and CAIX protein using the proximity ligation assay. Analysis was performed in C33a cells transiently transfected with full length CAIX-wt, CAIX-A459G or mock control and cultured in hypoxia for an additional 48 h. The green PLA signal indicating the interaction of CAIX with PIMT was clearly visible in the cells expressing wild type CAIX, while in CAIX-A459G the extent and intensity of the signal was considerably lower. C33a-mock transfectants served as a negative assay control due to a lack of one target protein. CAIX protein was post-labelled in red, nuclei are blue. Images were taken using objective 40× and zoom 3.

Article Snippet: Samples were blocked with 3% BSA/PBS for 30 min, incubated with a mixture of antibodies against CAIX (NB100-417, Novus Biological, Centennial, Colorado; diluted to 1 µg/mL) and PIMT (sc-100977, Santa Cruz Biotechnology, Dallas, TX, USA; diluted to 2 µg/mL) or CAIX (M75, diluted to 1 µg/mL) and AE2 (GenScript, Piscataway, NJ, USA; diluted to 1 µg/mL) for 1 h, incubated with plus and minus PLA probes for 1 h, then incubated with a ligation mixture containing connector oligonucleotides for 30 min, and finally with an amplification mixture containing a fluorescently labelled DNA probe for 100 min.

Techniques: Staining, Purification, SDS Page, Western Blot, Negative Control, In Situ, Proximity Ligation Assay, Transfection, Cell Culture, Expressing

Journal: International Journal of Molecular Sciences

Article Title: PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity

doi: 10.3390/ijms21228545

Figure Lengend Snippet: Cancer-associated expression of Pcmt1 /PIMT: ( a ) Output from GEPIA analysis of Pcmt1 expression in healthy versus tumor tissues ( http://gepia.cancer-pku.cn/about.html ) . GEPIA is a web server for analyzing the RNA sequencing expression data of 9736 tumors and 8587 normal samples from the TCGA and the GTEx projects, using a standard processing pipeline. Increased tumor-associated Pcmt1 levels were shown in a wide spectrum of cancer-types: BRCA-breast invasive carcinoma, CESC-cervical squamous cell carcinoma and endocervical adenocarcinoma, COAD-colon adenocarcinoma, ESCA-esophageal carcinoma, HNSC-head and neck squamous adenocarcinoma, LIHC-liver hepatocellular carcinoma, LUSC-lung squamous cell carcinoma, PAAD-pancreatic adenocarcinoma, PRAD-prostate adenocarcinoma, READ-rectum adenocarcinoma, STAD-stomach adenocarcinoma, UCS-uterine carcinosarcoma. Data were expressed in Log2 scale, n = number of samples. ( b ) Output from GENEVESTIGATOR analysis of Pcmt1 and CA9 gene expression levels in various cancer types ( https://genevestigator.com/ ). Data were selected from HS_AFFY_U133PLUS_2-0, analyzed using Cancer/ScatterplotTree settings and expressed as Log2. ( c ) Representative image of immunohistochemical staining of CAIX and PIMT in CRC patient tissue sample. Images were taken using objective 40×. ( d ) Representative image showing the interaction between PIMT and CAIX protein in CRC patient tissue samples detected by proximity ligation assay. Red PLA signals indicating the interaction of CAIX with PIMT, nuclei are blue. Negative control was prepared by omission of the PIMT primary antibody. Images were taken using objective 40× and zoom 3.

Article Snippet: Samples were blocked with 3% BSA/PBS for 30 min, incubated with a mixture of antibodies against CAIX (NB100-417, Novus Biological, Centennial, Colorado; diluted to 1 µg/mL) and PIMT (sc-100977, Santa Cruz Biotechnology, Dallas, TX, USA; diluted to 2 µg/mL) or CAIX (M75, diluted to 1 µg/mL) and AE2 (GenScript, Piscataway, NJ, USA; diluted to 1 µg/mL) for 1 h, incubated with plus and minus PLA probes for 1 h, then incubated with a ligation mixture containing connector oligonucleotides for 30 min, and finally with an amplification mixture containing a fluorescently labelled DNA probe for 100 min.

Techniques: Expressing, RNA Sequencing Assay, Immunohistochemical staining, Staining, Proximity Ligation Assay, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity

doi: 10.3390/ijms21228545

Figure Lengend Snippet: Functional role of PIMT binding to CAIX: ( a ) Effect of Pcmt1 transient silencing on CAIX-mediated extracellular acidification. The graph shows the differences between pH values (ΔpH) measured in culture medium at the beginning and after 48 h in hypoxia normalized to protein concentration. Results clearly showed that transient Pcmt1 silencing in C33a-CAIX cells (stable CAIX transfectants) resulted in the alkalinization of extracellular pHe when compared to scramble control (scr). Measurements were performed after 72 h post-transfection of siRNAs. The data are presented as the mean ± s.d., n = 6. Statistical significance was analyzed using the Student’s t -test and expressed as a p -value (* p < 0.05). ( b ) Efficacy of transient silencing was checked in parallel at the protein level using immunoblot analysis depicted on a representative image. ( c ) Activation of CAIX enzymatic function under hypoxic conditions. Posttranslational modification of the intracellular tail is necessary for full activation of CAIX enzymatic function. Phosphorylation of Thr443 was identified previously. In this work we found out a novel condition necessary for CAIX activation—interaction between the IC tail of CAIX and PIMT facilitated by terminal Ala459. Hypoxia can affect the activity of CAIX in several ways: it upregulates cAMP levels through ADCY6 and ADCY7, leading to higher levels of cAMP, PKA activation and Thr443 phosphorylation, and also enhances the expression of PIMT, allowing its interaction with the IC tail of CAIX.

Article Snippet: Samples were blocked with 3% BSA/PBS for 30 min, incubated with a mixture of antibodies against CAIX (NB100-417, Novus Biological, Centennial, Colorado; diluted to 1 µg/mL) and PIMT (sc-100977, Santa Cruz Biotechnology, Dallas, TX, USA; diluted to 2 µg/mL) or CAIX (M75, diluted to 1 µg/mL) and AE2 (GenScript, Piscataway, NJ, USA; diluted to 1 µg/mL) for 1 h, incubated with plus and minus PLA probes for 1 h, then incubated with a ligation mixture containing connector oligonucleotides for 30 min, and finally with an amplification mixture containing a fluorescently labelled DNA probe for 100 min.

Techniques: Functional Assay, Binding Assay, Protein Concentration, Transfection, Western Blot, Activation Assay, Modification, Activity Assay, Expressing

Journal: Cancers

Article Title: Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts

doi: 10.3390/cancers11071002

Figure Lengend Snippet: Sunitinib induces hypoxia and Carbonic Anhydrase IX (CAIX) expression in primary Triple Negative Breast Cancer (TNBC) tumors. ( a ) Images of MDA-MB-231 LM2-4Luc + primary tumor tissue sections harvested at increasing tumor volumes from animals administered either vehicle or 60 mg/kg sunitinib and immunohistochemically stained for the indicated markers. Scale bars: upper panels, 1 mm; lower panels, 200 μm. ( b to e ) Image-based quantification of ( b ) CD31 + blood vessels, ( c ) CAIX expression, ( d ) pimonidazole and ( e ) proliferation. Data show the mean ± standard error of the mean (SEM). n = 3 animals/group with 10 images/animal. * p < 0.05, *** p < 0.001.

Article Snippet: Formalin-fixed, paraffin-embedded (FFPE) tissues were sectioned (4–5 μm) and stained with primary antibodies against human CAIX (1:50, AF2188; R&D Systems, Minneapolis, MN, USA), CD31 (1:10, DIA-310; Histobiotec, Miami Beach, FL, USA), BrdU (1:10, Roche, Laval, QC, Canada) and pimonidazole (1:100, Hydroxyprobe, Burlington, MA, USA).

Techniques: Expressing, Staining

Journal: Cancers

Article Title: Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts

doi: 10.3390/cancers11071002

Figure Lengend Snippet: SLC-0111 reduces vessel density and vascular permeability in primary tumors, and decreases lung and liver metastases. ( a ) Representative 3D maximum projections of whole mount primary tumor tissue slices showing CD31 + blood vessels (green) and fluorescently labeled dextran (red). Merged images demonstrate the presence of both intravascular (yellow) and extravasated (arrowheads) dextran. Scale bar = 100 μm. ( b ) Quantification of the number of vessels in whole mount primary tumor slices. Data show the mean ± standard error of the mean (SEM). n = 13–14 images/group. ** p < 0.01, *** p < 0.001. ( c ) Quantification of vascular permeability as assessed by relative area of extravasated dextran in whole mount primary tumor slices. Data show the mean ± SEM. n = 13–16 images/group. * p < 0.05, ** p < 0.01. ( d ) Representative 3D maximum projections of whole mount primary tumor tissue slices showing levels of CAIX expression (green) and the number of CD31 + blood vessels (red). Lower panels, merge. Scale bar = 100 μm. ( e ) Quantification of Carbonic Anhydrase IX (CAIX) expression in whole mount primary tumor tissue slices in panel d. Data show the mean ± SEM. n = 11–15 images/group. * p < 0.05, *** p < 0.001. ( f ) Representative images of lung and liver tissues from animals with MDA-MB-231 LM2-4Luc + orthotopic breast tumors and administered SLC-0111 and sunitinib, either alone or in combination. Visible metastatic nodules (arrows) are indicated. Scale bar = 1 cm. ( g – i ) Analysis of ( g ) lung weight ( n = 9/group), ( h ) liver weight as a function of whole animal body weight ( n = 9/group) and ( i ) number of metastatic nodules present on the liver surface ( n = 7–8/group). For each graph, data show the mean ± SEM. * p < 0.05.

Article Snippet: Formalin-fixed, paraffin-embedded (FFPE) tissues were sectioned (4–5 μm) and stained with primary antibodies against human CAIX (1:50, AF2188; R&D Systems, Minneapolis, MN, USA), CD31 (1:10, DIA-310; Histobiotec, Miami Beach, FL, USA), BrdU (1:10, Roche, Laval, QC, Canada) and pimonidazole (1:100, Hydroxyprobe, Burlington, MA, USA).

Techniques: Permeability, Labeling, Expressing

Journal: Cancers

Article Title: Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts

doi: 10.3390/cancers11071002

Figure Lengend Snippet: Administration of SLC-0111 reduces lung metastases. ( a ) Representative 3D maximum projections of whole mount lung tissue slices from tumor-bearing mice, showing vimentin-positive metastases (cyan) and CD31 + blood vessels (red). Scale bar = 100 μm. ( b ) Analysis of vimentin positive metastases in whole mount lung tissue sections described in panel a. Data show the mean ± standard error of the mean (SEM). n = 8–10 images/group. * p < 0.05, *** p < 0.001. ( c ) Representative 3D maximum projections of whole mount lung tissue slices from tumor-bearing mice, showing Carbonic Anhydrase IX (CAIX)-positive metastases (green) and CD31 + blood vessels (red). Bar = 100 μm. ( d ) Analysis of CAIX positive metastases in the whole mount lung tissue sections described in panel c. Data show the mean ± SEM. n = 5–8 images/group: No statistically significant differences were observed among the groups.

Article Snippet: Formalin-fixed, paraffin-embedded (FFPE) tissues were sectioned (4–5 μm) and stained with primary antibodies against human CAIX (1:50, AF2188; R&D Systems, Minneapolis, MN, USA), CD31 (1:10, DIA-310; Histobiotec, Miami Beach, FL, USA), BrdU (1:10, Roche, Laval, QC, Canada) and pimonidazole (1:100, Hydroxyprobe, Burlington, MA, USA).

Techniques:

Journal: Cancers

Article Title: Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts

doi: 10.3390/cancers11071002

Figure Lengend Snippet: Model for targeting angiogenesis and Carbonic Anhydrase IX (CAIX) to reduce metastasis of Triple Negative Breast Cancer (TNBC). Exposure of tumors to anti-angiogenic agents such as sunitinib leads to reduced tumor growth. However, major consequences can include enhanced hypoxia and increased vascular permeability. The exacerbation of hypoxia results in the upregulation of CAIX expression by tumor cells and the potentiation of metastasis. Inhibition of CAIX activity inhibits metastasis through several mechanisms, as shown by previous studies, but may also play a role in reducing vascular permeability and contributing to vascular normalization, potentially reducing the invasion of tumor cells to distant sites.

Article Snippet: Formalin-fixed, paraffin-embedded (FFPE) tissues were sectioned (4–5 μm) and stained with primary antibodies against human CAIX (1:50, AF2188; R&D Systems, Minneapolis, MN, USA), CD31 (1:10, DIA-310; Histobiotec, Miami Beach, FL, USA), BrdU (1:10, Roche, Laval, QC, Canada) and pimonidazole (1:100, Hydroxyprobe, Burlington, MA, USA).

Techniques: Permeability, Expressing, Inhibition, Activity Assay

Journal:

Article Title: Role of Hypoxia and EGF on Expression, Activity, Localization and Phosphorylation of Carbonic Anhydrase IX in MDA-MB-231 Breast Cancer Cells

doi: 10.1016/j.bbamcr.2010.09.018

Figure Lengend Snippet: MDA-MB-231 cells were exposed to hypoxia or not for 16 h in the absence of serum. EGF (16nM) was added for 30 min after which total membranes were isolated. CAIX was immunoprecipitated with an antibody generated in goat (R&D Systems, #AF2188) followed by western blotting with an anti-phosphotyrosine antibody or the M75mouse monoclonal antibody. C = control, H = hypoxia. These data represent triplicate experiments.

Article Snippet: The beads were removed by centrifugation at 3000, × g for 1 min. To the precleared supernatants, 50 μL of a 50% solution of protein A/G plus-agarose beads and 3 μg of a (goat) polyclonal antibody against CAIX (R&D Systems #AF2188) were added and subjected to gentle end-over-end mixing overnight at 4° C. The immune complexes were collected by centrifugation at 3000 × g for 5 min. Immunoprecipitates were subjected to gel electrophoresis and blotted onto nitrocellulose membranes.

Techniques: Isolation, Immunoprecipitation, Generated, Western Blot