rabbit polyclonal anti caix antibody nb100 417  (Novus Biologicals)

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (A) Amino acid sequence of the human CAIX proteoglycan-like domain. Negatively charged amino acids are labelled in red, positively charged amino acids are labelled in blue. (B, C) Original recordings of the change in intracellular H + concentration ([H + ] i ) in oocytes expressing (B) MCT1 or (C) MCT4 (black trace), MCT1/4 + CAIX-WT (blue trace), and MCT1/4 + CAIX-ΔPG (red trace), respectively, during application of 3 and 10 mM of lactate and 5% CO 2 / 10 mM HCO 3 - . (D-I) Rate of change in intracellular H + concentration (Δ[H + ]/Δt) as induced by application (D, G) and removal (E, H) of 3 and 10 mM lactate, and application of 5% CO 2 / 10 mM HCO 3 - (F, I), respectively, in oocytes expressing MCT1/4 (gray), MCT1/4+CAIX-WT (blue), and MCT1+CAII-ΔPG (red), respectively. Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars for MCT1/4+CAIX/CAIX-ΔPG-coexpressing oocytes refer to the values from oocytes expressing MCT1/4 alone. The blue significance indicators above the bars for MCT1/4+CAIX-ΔPG-coexpressing oocytes refer to the values from oocytes coexpressing MCT1/4 + CAIX.

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Sequencing, Concentration Assay, Expressing, Comparison

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: Antibody staining of fixed and permeabilized oocytes, expressing MCT1+CAIX-WT (A) , MCT4+CAIX-WT (B) , MCT1+CAIX-ΔPG (C) , MCT4+CAIX-ΔPG (D) , and a native oocyte as control (E) . CAIX was labeled with an antibody, mapping against the C-terminal region of CAIX. Pictures were taken with a confocal laser scanning microscope.

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Staining, Expressing, Control, Labeling, Laser-Scanning Microscopy

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (A, B) Original recordings of the change in intracellular H + concentration ([H + ] i ) in oocytes coexpressing MCT1+CAIX-WT (A) and MCT4+CAIX (B), respectively, during application of 3 and 10 mM lactate. Cells were pre-incubated for 24 h with 0.4 μg/ml Anti-PG (red traces), 0.4 μg/ml Anti-CA (green traces) or without antibodies (black traces) before the measurements were carried out. (C, D) Rate of change in intracellular H + concentration (Δ[H + ]/Δt) as induced by application (C, E) and removal (D, F) of 3 and 10 mM lactate, in oocytes expressing MCT1/4 (gray) and MCT1/4+CAIX-WT (blue), respectively, in the presence of Anti-PG, Anti-CA or in the absence of antibodies (control). Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars for MCT1/4+CAIX-coexpressing oocytes refer to the corresponding values from oocytes expressing MCT1/4 alone (gray bars). The blue significance indicators above the bars for MCT1/4+CAIX-coexpressing oocytes, incubated with Anti-PG or Anti-CA refer to the values from MCT1/4+CAIX-coexpressing oocytes not incubated with antibody (blue bars). No significant changes were found between MCT1/4-expressing oocytes incubated with or without antibodies (gray bars).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Concentration Assay, Incubation, Expressing, Control, Comparison

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (A) Original recordings of the log enrichment (LE), as measured by gas-analysis mass spectrometry, of a pool of 20 lysed, native oocytes (black trace) and pools of 20 lysed oocytes, expressing either CAIX-WT (blue trace) or CAIX-ΔPG (red trace). The beginning of the traces shows the rate of degradation of the 18 O-labeled substrate in the non-catalyzed reaction. The black arrowhead indicates addition of oocyte lysate. (B) Enzymatic activity of lysates from 20 native oocytes (gray) and 20 oocytes expressing either CAIX-WT (blue) or CAIX-ΔPG (red), respectively. One unit is defined as 100% stimulation of the non-catalyzed 18 O depletion of doubly labelled 13 C 18 O 2 . Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars from CAIX-expressing oocytes refer to the values from native cells (gray bar). The blue asterisks above the bar from CAIX-ΔPG-expressing oocytes refer to the values from CAIX-WT-expressing oocytes (blue bar). (C) Enzymatic activity of cell lysate from 20 native (gray) and CAIX-expressing oocytes (blue, red and green), respectively. Cell lysates were incubated for 2h with 5 μg/ml of Anti-PG (red), 5 μg/ml of Anti-CA (green), or without antibody (blue). One unit is defined as 100% stimulation of the non-catalyzed 18 O depletion of doubly labelled 13 C 18 O 2 . Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars from CAIX-expressing oocytes refer to the values from native cells (gray bar). The blue asterisks above the bar from CAIX-expressing oocytes, incubated with antibodies refer to the values from CAIX-WT-expressing oocytes without antibody (blue bar). The red asterisks above the bar from CAIX-expressing oocytes, incubated with Anti-CA refer to the values from CAIX-WT-expressing oocytes, incubated with Anti-PG (red bar).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Mass Spectrometry, Expressing, Labeling, Activity Assay, Comparison, Incubation

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (A) Original recordings of the change in intracellular pH (pH i ) during application of 3 and 10 mM of lactate in MCF-7 breast cancer cells, incubated under normoxia (left traces) or hypoxia (right traces), in the presence of 5 μg/ml Anti-PG (red traces), 5 μg/ml Anti-CA (green traces) or in the absence of antibodies (black traces). (B, C) Rate of change in intracellular pH, as induced by application (B) and removal (C) of lactate in MCF-7 cells, incubated under normoxia (gray) or hypoxia (blue), in the presence of Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). (D) Relative change in intracellular lactate concentration, as induced by application of lactate in MCF-7 cells, incubated under normoxia (gray) or hypoxia (blue) in the presence of Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). Data are represented as mean ± SEM. Significance in differences was tested with ANOVA, followed by means comparison. The significance indicators above the bars from cells, incubated with antibody refer to the corresponding values from control cells (Ctrl). (E) CA catalytic activity of MCF-7 cells, as measured by gas-analysis mass spectrometry, in the presence 5 μg/ml Anti-PG (red), 5 μg/ml Anti-CA (green), 30 μM of the CA inhibitor EZA (yellow) and in the absence of antibodies or inhibitors (Ctrl). Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black asterisks above the bars from cells treated with antibodies or EZA refer to the values from control cells (gray bar). The red asterisks above the bars from cells treated with Anti-CA or EZA refer to the values from cells treated with Anti-PG (red bar). The green significance indicator above the bar from cells treated with EZA refers to the values from cells treated with Anti-CA (green bar). (F) Western blot against CAIX and β-tubulin as loading control, in MCF-7 cells, incubated in the presence Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). (G) Quantification of the protein level of CAIX in MCF-7 cells, incubated in the presence Anti-PG (red), Anti-CA (green) or in the absence of antibodies (gray) relative to the protein level of β-tubulin. Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars from cells, incubated with antibody refer to the corresponding values from control cells (gray bar). The red significance indicator above the bar from cells treated with Anti-CA refers to the values from cells treated with Anti-PG (red bar).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Incubation, Concentration Assay, Comparison, Control, Activity Assay, Mass Spectrometry, Western Blot

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (A) Original recordings of the change in intracellular pH (pH i ) during application of 3 and 10 mM of lactate, in MDA-MB-231 breast cancer cells, incubated under normoxia (left traces) or hypoxia (right traces), in the presence of 5 μg/ml Anti-PG (red traces), 5 μg/ml Anti-CA (green traces) or in the absence of antibodies (black traces). (B, C) Rate of change in intracellular pH, as induced by application (B) and removal (C) of lactate in MDA-MB-231 cells, incubated under normoxia (gray) or hypoxia (blue), in the presence of Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). (D) Relative change in intracellular lactate concentration, as induced by application of lactate in MDA-MB-231 cells, incubated under normoxia (gray) or hypoxia (blue) in the presence of Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). Data are represented as mean ± SEM. Significance in differences was tested with ANOVA, followed by means comparison. The significance indicators above the bars from cells, incubated with antibody refer to the corresponding values from control cells (Ctrl). (E) CA catalytic activity of MDA-MB-231 cells, as measured by gas-analysis mass spectrometry, in the presence 5 μg/ml Anti-PG (red), 5 μg/ml Anti-CA (green), 30 μM of the CA inhibitor EZA (yellow) and in the absence of antibodies or inhibitors (Ctrl). Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black asterisks above the bars from cells treated with antibodies or EZA refer to the values from control cells (gray bar). The red asterisks above the bars from cells treated with Anti-CA or EZA refer to the values from cells treated with Anti-PG (red bar). The green significance indicator above the bar from cells treated with EZA refers to the values from cells treated with Anti-CA (green bar). (F) Western blot against CAIX and β-tubulin as loading control, in MDA-MB-231 cells, incubated in the presence Anti-PG, Anti-CA or in the absence of antibodies (Ctrl). (G) Quantification of the protein level of CAIX in MDA-MB-231 cells, incubated in the presence Anti-PG (red), Anti-CA (green) or in the absence of antibodies (gray) relative to the protein level of β-tubulin. Data are represented as mean + SEM. Significance in differences was tested with ANOVA, followed by means comparison. The black significance indicators above the bars from cells, incubated with antibody refer to the corresponding values from control cells (gray bar). The red significance indicator above the bar from cells treated with Anti-CA refers to the values from cells treated with Anti-PG (red bar).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Incubation, Concentration Assay, Comparison, Control, Activity Assay, Mass Spectrometry, Western Blot

Journal: Oncotarget

Article Title: The proteoglycan-like domain of carbonic anhydrase IX mediates non-catalytic facilitation of lactate transport in cancer cells

doi: 10.18632/oncotarget.25371

Figure Lengend Snippet: (A) Western blot of CAIX, in normoxic and hypoxic MCF-7 and MDA-MB-231 cells with Anti-PG (Ab-M75, upper blots). β-tubulin was used as loading control (lower blots). (B) Quantification of the protein level of CAIX in normoxic and hypoxic MDA-MB-231 and MCF-7 cells, respectively, relative to the protein level of β-tubulin. Data are represented as mean + SEM. Significance in differences was tested with Student's t-test, following a Shapiro-Wilk test. The significance indicators above the bars for normoxic and hypoxic MCF-7 cells refer to the corresponding values for normoxic and hypoxic MDA-MB-231 cells (gray bars).

Article Snippet: Detection of CAIX was performed either with rabbit anti-human CAIX polyclonal antibody (2 μg/ml; NB100-417, Novus Biologicals), for samples of cells treated with anti-CAIX monoclonal antibodies, or with mouse anti-human CAIX monoclonal antibody clone Anti-PG (M75; 0.4 μg/ml).

Techniques: Western Blot, Control

Journal: Journal of Biomarkers

Article Title: A Sensitive IHC Method for Monitoring Autophagy-Specific Markers in Human Tumor Xenografts

doi: 10.1155/2016/1274603

Figure Lengend Snippet: Comparison of LC3B levels in CAIX-positive (hypoxic) and CAIX-negative (normoxic) tumor areas. (a) H&E images Calu-6 xenograft tumor sections from vehicle-treated nude mice. (b) Standard IHC double labeling of CAIX (red) and pS6 (brown) in Calu-6 xenograft tumor sections from vehicle-treated nude mice. CAIX-positive staining (red stain) delineating hypoxic conditions was predominately observed in viable tumor adjacent to necrotic tumor areas (red stain), while the mTOR pathway marker pS6 showed positivity (brown stain) predominately in the normoxic region of tumor determined by CAIX-negative staining. There was very little overlap between the CAIX and pS6 marker staining. (c) Labeling of CAIX and LC3B (left image) was performed on separate Calu-6 xenograft serial sections to annotate CAIX-positive stained areas as hypoxic (H) and CAIX-negative areas as normoxic (N). CAIX-positive and CAIX-negative regions of the tumor sections were manually annotated on to LC3B stained slides indicated with blue lines. Inner circles within the viable tumor delineate areas with no CAIX staining were labeled as normoxic (N). Viable tumor positive for CAIX staining was labeled as hypoxic (H). (d) LC3B positive pixel image analysis results comparing hypoxic (H) regions with normoxic (N) tumor regions showed a significant difference in LC3B level between the two regions ( p value = 0.012).

Article Snippet: The following primary antibodies were used: polyclonal rabbit anti-carbonic anhydrase IX (CAIX) antibody (Novus Biologicals, NB100-417, 1 : 2500); polyclonal rabbit anti-SQSTM1/P62 antibody (Abcam, 91526, 1 : 5000); polyclonal rabbit anti-ATG7 (Epitomics, 2054-1, 1 : 800); monoclonal rabbit anti-pS6 (Cell Signaling Technology, 5364, 1 : 2500); monoclonal rabbit anti-LC3B (Cell Signaling Technology, 3868, 1 : 50); and monoclonal rabbit anti-NBR1 (inhouse antibody, 1 : 50; validation of ML85-61-3 data not shown).

Techniques: Comparison, Labeling, Staining, Marker, Negative Staining

Journal: Scientific Reports

Article Title: Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function

doi: 10.1038/srep13605

Figure Lengend Snippet: ( a ) Determination of the K m value for lactate in MCF-7 cells under normoxic (21% O 2 , grey) and hypoxic (1% O 2 , blue) conditions, respectively, as determined by the rate of change in pH i during application of 0.3, 1, 3, 10 and 30 mM lactate. Western blots of lysate from MCF-7 cells, incubated under normoxic (21% O 2 ) and hypoxic (1% O 2 ) conditions, labelled for MCT1 ( b ), MCT2 ( c ) and MCT4 ( d ), respectively. For positive control of MCT2, lysate from MCT2-expressing oocytes was used. Actin was used as loading control. ( e ) Relative change in the RNA level of MCT1 and MCT4 in MCF-7 cells after three days under hypoxic conditions. ( f ) Relative change in the RNA level of NHE1 and NBCn1 in MCF-7 cells after three days under hypoxic conditions. ( g ) Relative change in the RNA level of CAII and CAIX in MCF-7 cells after three days under hypoxic conditions. Expression level of CAIX is strongly upregulated, while the expression levels of MCT1, MCT4, NHE1 and NBCe1 show no significant changes. ( h ) Western blot of MCF-7 cell lysate, labelled for CAIX and actin as loading control. ( i ) Quantification of CAIX protein level by western blot analysis in MCF-7 cells under normoxic (21% O 2 ) and hypoxic (1% O 2 ) conditions, respectively. ( j ) Western blots of lysate from MCF-7 cells, incubated under normoxic (21% O 2 ) and hypoxic (1% O 2 ) conditions, labelled for NHE1 and actin. Data are represented as mean ± SEM.

Article Snippet: Cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) (Roti-Histofix ® 4%, Carl Roth GmbH + Co. KG) for 15 min. After blocking of unspecific binding sites with blocking solution (1:1000 Triton X-100, 1% goat serum, 3% bovine serum albumin (BSA)) for 60 min, cells were incubated with CAIX rabbit anti-human CAIX polyclonal antibody (diluted to 1:500; NB100-417, Novus Biologicals Ltd., UK) for 2 hours.

Techniques: Western Blot, Incubation, Positive Control, Expressing, Control

Journal: Scientific Reports

Article Title: Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function

doi: 10.1038/srep13605

Figure Lengend Snippet: ( a ) Antibody staining for CAIX (green) in MCF-7 cells, kept under hypoxic conditions. Hypoxic cells either remained untreated (a1), mock-transfected with non-targeting negative control siRNA (a2) or transfected with siRNA against CAIX (a3). Nuclei are stained with Hoechst (blue). ( b ) Quantification of the fluorescent signal for CAIX as shown in (a). ( c ) Original recording of the relative change in intracellular lactate concentration in MCF-7 cells kept under hypoxic conditions during application of 1 and 3 mM lactate. Cells were either untreated (black trace), mock-transfected with non-targeting negative control siRNA (green trace) or transfected with siRNA against CAIX (blue trace). ( d ) Rate of change in lactate level during application of 1 and 3 mM lactate in hypoxic MCF-7 cells, either untreated (gray bars), mock-transfected with non-targeting negative control siRNA (green bars) or transfected with siRNA against CAIX (blue bars). Knock-down of CAIX induced a significant decrease in lactate flux. ( e ) Original recordings of changes in pH i in hypoxic MCF-7 cells, either untreated (control, gray traces), mock-transfected with non-targeting negative control siRNA (green traces) or transfected with siRNA against CAIX (blue traces). ( f , g ) Rate of change in pH i , as induced by application ( f ) and removal ( g ) of lactate, respectively. ( h ) Original recordings of the relative change in intracellular lactate concentration in MCF-7 cells kept under normoxic (blue traces) or hypoxic (red traces) conditions during application of lactate in the presence and absence of 5% CO 2 /15 mM HCO 3 − and 30 μM EZA, respectively. ( i ) Rate of change in intracellular lactate concentration in MCF-7 cells under normoxic and hypoxic conditions, respectively, as induced by application of 1 mM lactate in the absence and presence of 5% CO 2 /15 mM HCO 3 − and 30 μM EZA, respectively. Hypoxia induces a significant increase in lactate flux both in the absence and in the presence of CO 2 /HCO 3 − and EZA. Knockdown of CAIX induced a significant decrease in the rate of change in pH i , both during addition and removal of lactate. Data are represented as mean ± SEM.

Article Snippet: Cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) (Roti-Histofix ® 4%, Carl Roth GmbH + Co. KG) for 15 min. After blocking of unspecific binding sites with blocking solution (1:1000 Triton X-100, 1% goat serum, 3% bovine serum albumin (BSA)) for 60 min, cells were incubated with CAIX rabbit anti-human CAIX polyclonal antibody (diluted to 1:500; NB100-417, Novus Biologicals Ltd., UK) for 2 hours.

Techniques: Staining, Transfection, Negative Control, Concentration Assay, Knockdown, Control

Journal: Scientific Reports

Article Title: Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function

doi: 10.1038/srep13605

Figure Lengend Snippet: ( a ) Original recordings of intracellular H + -concentration ([H + ] i ) in Xenopus oocytes expressing MCT1 (black trace), MCT1+CAIX-WT (blue trace), or MCT1+CAIX-H200A (green trace), respectively, during application of 3 and 10 mM lactate and of 5% CO 2 /10 mM HCO 3 − . ( b , c ) Rate of rise in [H + ] i , as induced by application of lactate ( b ) or 5% CO 2 /10 mM HCO 3 − ( c ) in oocytes expressing MCT1, MCT1+CAIX-WT and MCT1+CAIX-H200A, respectively. ( d ) Original recordings of the log enrichment of 20 native oocytes and 20 oocytes expressing either CAIX-WT or CAIX-H200A. The beginning of the traces shows the rate of degradation of the 18 O-labeled substrate in the non-catalysed reaction. The black arrowhead indicates addition of oocytes. ( e ) Enzymatic activity of native oocytes and oocytes expressing either CAIX-WT or CAIX-H200A. One unit is defined as 100% stimulation of the non-catalysed 18 O depletion of doubly labelled 13 C 18 O 2 . Data are represented as mean ± SEM.

Article Snippet: Cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) (Roti-Histofix ® 4%, Carl Roth GmbH + Co. KG) for 15 min. After blocking of unspecific binding sites with blocking solution (1:1000 Triton X-100, 1% goat serum, 3% bovine serum albumin (BSA)) for 60 min, cells were incubated with CAIX rabbit anti-human CAIX polyclonal antibody (diluted to 1:500; NB100-417, Novus Biologicals Ltd., UK) for 2 hours.

Techniques: Concentration Assay, Expressing, Labeling, Activity Assay

Journal: Scientific Reports

Article Title: Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function

doi: 10.1038/srep13605

Figure Lengend Snippet: ( a ) Staining of nuclei with Hoechst (blue) in MCF-7 cells after 3 days in culture. Hypoxic cells remained either untreated (a1), mock-transfected with non-targeting negative control siRNA (a2), transfected with siRNA against CAIX (a3), incubated with the CA inhibitor EZA (a4), or incubated with the MCT1 inhibitor AR-C155858 (a5). ( b ) Total number of nuclei/mm 2 in MCF-7 cell cultures, kept for 0–3 days under the conditions as described in ( a ). For every data point four dishes of cells were used and five pictures were taken from each dish at random locations, yielding 20 pictures/data point (n = 20/4). ( c ) Staining of dead MCF-7 cells with propidium iodide (red) after 3 days in culture. Living cells are visualised by phase contrast. Hypoxic cells remained either untreated (c1), mock-transfected with non-targeting negative control siRNA (c2), transfected with siRNA against CAIX (c3), incubated with the CA inhibitor EZA (c4), or incubated with the MCT1 inhibitor AR-C155858 (c5). As positive control, apoptosis was induced by application of staurosporine (c6). ( d ) Total number of living (grey) and dead (red) cells/mm 2 , kept for 3 days under the conditions as described in ( c ). For every data point 4 dishes of cells from two independent batches were used and 15 pictures were taken from each dish at random locations, yielding 60 pictures/data point (n =6 0/4). Data are represented as mean ± SEM.

Article Snippet: Cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) (Roti-Histofix ® 4%, Carl Roth GmbH + Co. KG) for 15 min. After blocking of unspecific binding sites with blocking solution (1:1000 Triton X-100, 1% goat serum, 3% bovine serum albumin (BSA)) for 60 min, cells were incubated with CAIX rabbit anti-human CAIX polyclonal antibody (diluted to 1:500; NB100-417, Novus Biologicals Ltd., UK) for 2 hours.

Techniques: Staining, Transfection, Negative Control, Incubation, Positive Control

Journal: Scientific Reports

Article Title: Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function

doi: 10.1038/srep13605

Figure Lengend Snippet: Under normoxic conditions (upper scheme), cancer cells rely on glycolysis and oxidative energy production in the tricarboxylic acid cycle (TCA) to meet their metabolic requirements. Under hypoxic conditions, glycolysis becomes the prime energy source, which leads to vast production of lactate (produced from pyruvate by lactate dehydrogenase, LDH) and H + . Under these conditions (lower left scheme), hypoxia-regulated CAIX, which is directly bound to the complex of MCT and its chaperon CD147, could move protons between the transporter pore and extracellular protonatable residues (light brown circles). Thereby CAIX can function as a ‘H + -distributing antenna’ for the MCT to facilitate rapid extrusion of lactate and H + from the cell. Knockdown of CAIX (lower right scheme) leads to loss of the ‘H + -distributing antenna’, which decreases MCT transport activity, leading to accumulation of lactate and H + in the cytosol. A detailed description of the mechanism is given in the Discussion section.

Article Snippet: Cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) (Roti-Histofix ® 4%, Carl Roth GmbH + Co. KG) for 15 min. After blocking of unspecific binding sites with blocking solution (1:1000 Triton X-100, 1% goat serum, 3% bovine serum albumin (BSA)) for 60 min, cells were incubated with CAIX rabbit anti-human CAIX polyclonal antibody (diluted to 1:500; NB100-417, Novus Biologicals Ltd., UK) for 2 hours.

Techniques: Produced, Knockdown, Activity Assay

Journal: BioMed Research International

Article Title: The Role of Hypoxia-Inducible Factor-1 α , Glucose Transporter-1, (GLUT-1) and Carbon Anhydrase IX in Endometrial Cancer Patients

doi: 10.1155/2014/616850

Figure Lengend Snippet: Immunohistochemical representative microphotographs representing the HIF-1 α , GLUT-1, and CAIX expression in endometrial cancer according to FIGO classification (IA, IB, II, IIIA, IIIC, and IV). Primary objective magnification 20x.

Article Snippet: The immunohistochemical studies were performed using, respectively, mouse monoclonal antibody against HIF-1 α (clone [H1ALPHA67], ab1, Abcam, Cambridge, UK), rabbit polyclonal antibody against GLUT-1 (07-1401, MILLOPORE), and rabbit polyclonal antibody against CAIX (NB100-417, NOVUS BIOLOGICALS, Cambridge, UK).

Techniques: Immunohistochemical staining, Expressing

Journal: BioMed Research International

Article Title: The Role of Hypoxia-Inducible Factor-1 α , Glucose Transporter-1, (GLUT-1) and Carbon Anhydrase IX in Endometrial Cancer Patients

doi: 10.1155/2014/616850

Figure Lengend Snippet: The CAIX expression according to clinicopathological features.

Article Snippet: The immunohistochemical studies were performed using, respectively, mouse monoclonal antibody against HIF-1 α (clone [H1ALPHA67], ab1, Abcam, Cambridge, UK), rabbit polyclonal antibody against GLUT-1 (07-1401, MILLOPORE), and rabbit polyclonal antibody against CAIX (NB100-417, NOVUS BIOLOGICALS, Cambridge, UK).

Techniques: Expressing, Standard Deviation

Journal: BioMed Research International

Article Title: The Role of Hypoxia-Inducible Factor-1 α , Glucose Transporter-1, (GLUT-1) and Carbon Anhydrase IX in Endometrial Cancer Patients

doi: 10.1155/2014/616850

Figure Lengend Snippet: Prognostic factors for overall survival selected by Cox's univariate analysis.

Article Snippet: The immunohistochemical studies were performed using, respectively, mouse monoclonal antibody against HIF-1 α (clone [H1ALPHA67], ab1, Abcam, Cambridge, UK), rabbit polyclonal antibody against GLUT-1 (07-1401, MILLOPORE), and rabbit polyclonal antibody against CAIX (NB100-417, NOVUS BIOLOGICALS, Cambridge, UK).

Techniques:

Journal: BioMed Research International

Article Title: The Role of Hypoxia-Inducible Factor-1 α , Glucose Transporter-1, (GLUT-1) and Carbon Anhydrase IX in Endometrial Cancer Patients

doi: 10.1155/2014/616850

Figure Lengend Snippet: Prognostic factors for overall survival selected by Cox's multivariate analysis.

Article Snippet: The immunohistochemical studies were performed using, respectively, mouse monoclonal antibody against HIF-1 α (clone [H1ALPHA67], ab1, Abcam, Cambridge, UK), rabbit polyclonal antibody against GLUT-1 (07-1401, MILLOPORE), and rabbit polyclonal antibody against CAIX (NB100-417, NOVUS BIOLOGICALS, Cambridge, UK).

Techniques: