Journal: Journal of Clinical Medicine

Article Title: Soluble Urokinase Plasminogen Activator Receptor as a Predictor of All-Cause Death in Patients Undergoing Coronary Angiography at 10-Year Follow-Up

doi: 10.3390/jcm13206158

Figure Lengend Snippet: Biochemical tests based on suPAR concentration.

Article Snippet: Deoxyribonuclease 1 (DNase1) was measured using the Human DNASE1 ELISA Kit (Novus Biologicals, a Bio-Techne brand, Abingdon, UK), and neutrophil elastase was quantified using the Human Neutrophil Elastase SimpleStep ELISA Kit (Abcam, Cambridge, UK), following similar procedures to those described above.

Techniques: Concentration Assay

Journal: Lupus Science & Medicine

Article Title: Truncated isoforms of GPSM2 containing the GoLoco motif region promote CD4 + T-cell migration in SLE

doi: 10.1136/lupus-2022-000742

Figure Lengend Snippet: A truncated isoform of GPSM2 promotes CD4 + T-cell migration in SLE. (A) Representative example of western blot analysis of GPSM2 and the truncated 35 kDa GPSM2 isoform in CD4 + T cells from patients with SLE (SLE) and HCs. GPSM2 overexpressing lysate served as a positive ctrl (+); no protein served as a negative ctrl (−). (B) Comparison of GPSM2 expression in CD4 + cells from patients with SLE (n=10) and healthy individuals (n=9). (C) Expression of the truncated GPSM2 isoform in CD4 + cells from patients with SLE (n=10) and healthy individuals (n=9). (D) Schematic view of the experimental set-up of an in vitro T-cell migration assay. (E) Inhibition of the truncated GPSM2 isoform with blocking antibodies (block) or ctrl antibodies leads to decreased migration of CD4+ T cells from patients with SLE (n=9). Data are presented as mean±SEM. Significance was calculated using unpaired (B, C) or paired (E) Student’s t-test. ctrl, control; GPSM2, G-protein signalling modulator 2; HC, healthy control; n.s., not significant.

Article Snippet: GPSM2 overexpressing lysate served as a positive control (Novus Biologicals, Centennial, USA; cat. no. NBL1-11310).

Techniques: Migration, Western Blot, Comparison, Expressing, In Vitro, Cell Migration Assay, Inhibition, Blocking Assay, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

doi: 10.1073/pnas.1501947112

Figure Lengend Snippet: Phylogeny and structure of C5orf30. (A) Maximum likelihood (ML) tree of C5orf30 generated using the human sequence as seed and a JTT evolutionary model by PhylomeDB. C5orf30 is set as an arbitrary root in the image. The tree shows identified orthologs by speciation events (blue nodes). The scale-bar for branch length is indicated. (B) Jpred secondary structure prediction for human C5orf30; box indicates poly-arginine stretch followed by serine residues (red cylinders, α-helices; green arrows, β-sheets).

Article Snippet: A C5orf30 overexpression lysate made from vector-transfected HEK293 cells was used as a positive control (Novus Biologicals).

Techniques: Generated, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

doi: 10.1073/pnas.1501947112

Figure Lengend Snippet: Representative multiple sequence alignment of C5orf30 protein sequences associated with the tree shown in Fig. 1. The amino acid residues are colored according to the CLUSTALX code (blue, hydrophobic; green, polar; magenta, acidic; red, basic; yellow, proline).

Article Snippet: A C5orf30 overexpression lysate made from vector-transfected HEK293 cells was used as a positive control (Novus Biologicals).

Techniques: Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

doi: 10.1073/pnas.1501947112

Figure Lengend Snippet: Tissue specific expression of C5orf30 in RA. C5orf30 mRNA expression in human tissues and cell lines are greater in RASF and myeloid derived cells from healthy volunteers (A). Quantitative expression of C5orf30 mRNA was detected at lower levels in PBLs from RA patients (n = 116) compared with healthy controls from the general population (n = 100). Individual results are expressed as the means and SEM of duplicate determinations (B). Immunohistochemistry reveals tissue-specific C5orf30 expression (C) in the synovial lining (D) and sublining layer (E). n = 5 control; n = 6 OA and RA. (F) C5orf30 colocalization with synoviocytes. The white arrows point to areas of colocalization (yellow) of C5orf30 (red) with RASF (green) surface protein or CD68-expressing macrophages (green). Colocalization was not evident with other cell types (G). (Scale bars: C, 100 μm; E, 50 μm.) n = 5 patients per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: A C5orf30 overexpression lysate made from vector-transfected HEK293 cells was used as a positive control (Novus Biologicals).

Techniques: Expressing, Derivative Assay, Immunohistochemistry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

doi: 10.1073/pnas.1501947112

Figure Lengend Snippet: Expression of C5orf30 at the mRNA level was determined in RASF cultures at passages (P) 3–8. There was no significant difference in expression of C5orf30 (A). RASF were treated for 4 h with TNF (50 ng/mL), hypoxia (HYP) (0.5% O2), and a combination of TNF and hypoxia. Cell apoptosis and necrosis was measured by flow cytometry where no significant difference was detected between groups (B). n = 3 patients, and the data are represented as means ± SD.

Article Snippet: A C5orf30 overexpression lysate made from vector-transfected HEK293 cells was used as a positive control (Novus Biologicals).

Techniques: Expressing, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

doi: 10.1073/pnas.1501947112

Figure Lengend Snippet: C5orf30 modulates the autoagressive phenotype of RASF. (A) C5orf30 mRNA expression in the TNF and hypoxia-treated samples are expressed relative to control untreated RASF (A). Hypoxia consistently induced an increased level of mRNA expression. TNF treatment reduced expression (0.5-fold), as did a combination of both TNF and hypoxia. n = 5 patients, and the data are represented as mean ± 95% confidence interval (dotted line represents change from untreated cells). This finding was confirmed by immunofluorescence (red, anti-C5orf30; blue, DAPI; green, LAMP-1) and Western blots (B). Protein lysates were collected from treated RASF, run on a Western blot, and probed for C5orf30 expression, using β-actin expression as a control. (The black line indicates discontinuous bands.) (B). In C5orf30KD studies, RASF were treated with 50 nM anti-C5orf30 siRNA or nontargeting (NTC) siRNA. C5orf30KD increased cell migration (C) and invasion compared with the NTC (D). Data are from n = 6 RASF donors. Volcano plot of microarray data are presented (E) and a pie chart showing differentially expressed genes based on cluster analysis (F). (Scale bars: 100 μm.)

Article Snippet: A C5orf30 overexpression lysate made from vector-transfected HEK293 cells was used as a positive control (Novus Biologicals).

Techniques: Expressing, Immunofluorescence, Western Blot, Migration, Microarray

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

doi: 10.1073/pnas.1501947112

Figure Lengend Snippet: RASF were treated with 50 nM anti-C5orf30 siRNA or 50 nM nontargeting (NTC) siRNA. (A) C5orf30 siRNA knockdown resulted in >70% loss of C5orf30 mRNA expression at 24 and 48 h after treatment. This loss of expression was also evident at the protein level as assessed by Western blotting (B) and immunofluorescence staining of RASF cultured on coverslips (C). Red is C5orf30, green is β-actin, and blue is the DAPI-stained nuclei.

Article Snippet: A C5orf30 overexpression lysate made from vector-transfected HEK293 cells was used as a positive control (Novus Biologicals).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

doi: 10.1073/pnas.1501947112

Figure Lengend Snippet: RASF were treated with 50 nM anti-C5orf30 siRNA or 50 nM nontargeting (NTC) siRNA. (A) Representative FACS dot plots and combined data from n = 6 RASF donors reveals C5orf30 siRNA knockdown did not significantly affect cell viability as assessed by annexin V/propidium iodide staining compared with NTC. (B) RASF proliferation was studied by 3H-thymidine incorporation after C5orf30KD. One curie of 3H-thymidine was added to the cultures during the last 24 h where it was clear that C5orf30KD did not affect cell proliferation. Paired samples were analyzed (n = 6) and statistically tested by using a paired two-tailed Student t test (P < 0.05) using GraphPad Prism 6.

Article Snippet: A C5orf30 overexpression lysate made from vector-transfected HEK293 cells was used as a positive control (Novus Biologicals).

Techniques: Staining, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

doi: 10.1073/pnas.1501947112

Figure Lengend Snippet: Hierarchical clustering of differentially expressed genes following C5orf30 KD in RASF

Article Snippet: A C5orf30 overexpression lysate made from vector-transfected HEK293 cells was used as a positive control (Novus Biologicals).

Techniques: Migration, Variant Assay, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

doi: 10.1073/pnas.1501947112

Figure Lengend Snippet: C5orf30 overexpression in RASF. cDNA prepared from transfected, mock-transfected, and untreated RASFs was loaded into wells of a 2% (wt/vol) agarose gel and run at 110 V for 90 min. Results indicate that transfection of C5orf30 into cells resulted in it being successfully overexpressed after 24 h. Representative bands are at the expected size for C5orf30 (160 bp). qRT-PCR confirmed the change in gene expression over the mock transfected to be >300-fold (A). Furthermore, results indicate that transfection was successful because the DDK tag is only expressed in C5orf30 and not mock-transfected cells 48 h after transfection (B). qRT-PCR was used to study changes in gene expression between the mock and C5orf30-transfected RASFs (C). Those genes that were shown to be down-regulated are shown to the left of the vertical dotted line, and those shown to be up-regulated are shown on the right. The horizontal dotted line represents no change from mock-transfected cells. The results from the PCR were analyzed by using a one-sample test with a theoretical mean of 1 (P < 0.01). Data are represented as means ± SEM, and experiments are from three independent RASF donors.

Article Snippet: A C5orf30 overexpression lysate made from vector-transfected HEK293 cells was used as a positive control (Novus Biologicals).

Techniques: Over Expression, Transfection, Agarose Gel Electrophoresis, Quantitative RT-PCR, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

doi: 10.1073/pnas.1501947112

Figure Lengend Snippet: C5orf30KD following in vivo administration was assessed in mouse tissue 72 h after injection of the siRNA complex. DBA/1 mice were injected i.v. with siRNA to C5orf30 and NTC (n = 3). After 3 d, the mice were culled and organs and joints assessed for knockdown by using qRT-PCR. The results show mRNA concentrations of C5orf30KD compared with the NTC-treated mice (A). The data were analyzed by using a one-sample Student t test comparing the gene of interest against theoretical mean of 1 using GraphPad Prism 6. Results show C5orf30 was significantly knocked down in the front and hind paws, liver, and spleen (P < 0.05). Next, the effect of C5orf30 knockdown on murine CIA was determined. Mice were treated with either C5orf30 siRNA, NTC siRNA (5 mg/kg), or transfection reagent (Vehicle), n = 10 mice per group. Control groups included mice with no disease (Healthy, n = 5) and n = 5 mice per group treated with dexamethasone (Dex) (0.5 mg/kg). This bone erosion was quantified by counting the average number of objects per slice on the 2D micro-CT images of the calcaneus, using image analysis software (B). Bars represent means + SD, and r = Spearman's rho, representing the correlations between the observed and predicted mean values for each treatment group. Representative images of front and hind paws (Top), MicroCT (Middle), and histological sections of H&E and Safronin ‘O’ stains (Bottom) are shown (C).

Article Snippet: A C5orf30 overexpression lysate made from vector-transfected HEK293 cells was used as a positive control (Novus Biologicals).

Techniques: In Vivo, Injection, Quantitative RT-PCR, Transfection, Micro-CT, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

doi: 10.1073/pnas.1501947112

Figure Lengend Snippet: C5orf30KD augments CIA. Mice were treated with C5orf30 siRNA, NTC siRNA (5 mg/kg), or transfection reagent (vehicle) n = 10 mice per group. A group of mice also received Dexamethasone (Dex) (0.5 mg/kg). C5orf30KD significantly increased arthritis (A). Representative front and hind paw images of CIA in mice treated with NTC or C5orf30 siRNA (B). At day 49, joint disease was assessed by MicroCT (C), H&E (D), and Safranin O (E). Analysis of C5orf30KD was performed byusing a one-way ANOVA with multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001). qRT-PCR of hind paws shows the relative mRNA expression of genes in the paws of C5orf30KD compared with NTC treated mice (F).

Article Snippet: A C5orf30 overexpression lysate made from vector-transfected HEK293 cells was used as a positive control (Novus Biologicals).

Techniques: Transfection, Quantitative RT-PCR, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: C5orf30 is a negative regulator of tissue damage in rheumatoid arthritis

doi: 10.1073/pnas.1501947112

Figure Lengend Snippet: We also measured the mRNA expression of C5orf30 in mouse joints and peripheral blood samples by qPCR at the end of the experiment (day 49). C5orf30 expression was significantly increased in the paws of mice in C5orf30KD over NTCKD, whereas this C5orf30 expression was reversed in the blood samples taken from the mice. The horizontal dotted line represents no change from NTC. n = 3 mice per group for gene expression studies, and n = 10 for C5orf30 expression. The results from the PCR were analyzed by using a one-sample test with a theoretical mean of 1 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: A C5orf30 overexpression lysate made from vector-transfected HEK293 cells was used as a positive control (Novus Biologicals).

Techniques: Expressing

Journal: BioMed Research International

Article Title: Glycosyltransferases as Markers for Early Tumorigenesis

doi: 10.1155/2015/792672

Figure Lengend Snippet: Antibodies used for immunohistochemical staining of breast cancer tissue samples.

Article Snippet: ST6GALNac1 , Rabbit , Polyclonal IgG , Novus Biologicals , Uterus , 1 : 500.

Techniques: Immunohistochemical staining, Staining, Positive Control

Journal: BioMed Research International

Article Title: Glycosyltransferases as Markers for Early Tumorigenesis

doi: 10.1155/2015/792672

Figure Lengend Snippet: Positive control (a) and isotype control (b). For positive control tissues certainly expressing the questionable antigens were stained. Placental tissue was used for GALNT6 staining, Colon tissue was used for GCNT2 and Uterus was chosen for ST6GALNac1 staining. Furthermore appropriate antibody concentrations were determined in the positive controls. For isotype control same tissues as for positive control are used, but primary antibody is replaced by a control serum, thus excluding unspecific binding signals of the primary antibody.

Article Snippet: ST6GALNac1 , Rabbit , Polyclonal IgG , Novus Biologicals , Uterus , 1 : 500.

Techniques: Positive Control, Control, Expressing, Staining, Binding Assay

Journal: BioMed Research International

Article Title: Glycosyltransferases as Markers for Early Tumorigenesis

doi: 10.1155/2015/792672

Figure Lengend Snippet: Staining of malignant breast tissue with antibodies against the three glycosyltransferases GALNT6 (a), GCNT2 (b), and ST6GALNac1 (c). Pictures were taken with different objectives (6,3x, 10x, 25x, and 40x; from left to right column) resulting in different magnifications of tissue structures.

Article Snippet: ST6GALNac1 , Rabbit , Polyclonal IgG , Novus Biologicals , Uterus , 1 : 500.

Techniques: Staining

Journal: BioMed Research International

Article Title: Glycosyltransferases as Markers for Early Tumorigenesis

doi: 10.1155/2015/792672

Figure Lengend Snippet: Statistical analysis of the investigated features. Statistically significant P values are seen for Her4 (all glycosyltransferases) and for tumour size (GALNT6; GCNT2 and ST6GALNac1 show borderline significance).

Article Snippet: ST6GALNac1 , Rabbit , Polyclonal IgG , Novus Biologicals , Uterus , 1 : 500.

Techniques:

Journal: Methods and Protocols

Article Title: Capillary Western Immunoassay Optimization of Estrogen Related Factors in Human Subcutaneous Adipose Tissue

doi: 10.3390/mps5020034

Figure Lengend Snippet: All electropherograms report lysate concentrations of 0.5 and 1.0 mg/mL with 1:10 and 1:50 antibody concentrations. These detection antibodies were not close to their target protein’s predicted molecular weight and were, thus, not considered to be specifically binding target proteins of interest: ( A ) HSD17ß1 ( B ) HSD17ß2 ( C ) HSD17ß4 ( D ) HSD17ß7 and ( E ) SULTe1.

Article Snippet: Control lysates tested include those for: HSD17ß1 (human placental whole cell lysate; NB820-59248, Novus Biologicals), HSD17ß2 (human embryonic kidney cells; NBL1-11726, Novus Biologicals), HSD17ß4 (human embryonic kidney cells; NBL1-11728, Novus Biologicals), HSD17ß7 (human embryonic kidney cells; NBP2-07042, Novus Biologicals) and SULTe1 (human embryonic kidney cells; ab94289, Abcam).

Techniques: Molecular Weight, Binding Assay

Journal: Methods and Protocols

Article Title: Capillary Western Immunoassay Optimization of Estrogen Related Factors in Human Subcutaneous Adipose Tissue

doi: 10.3390/mps5020034

Figure Lengend Snippet: Protein quantification in reduced lysate concentrations (0.0625, 0.125, 0.25, 0.3, 0.4, and 0.5 mg/mL) paired with identical antibody concentrations (1:10 and 1:50) for ( A ) HSD17ß2 and ( B ) HSD17ß7.

Article Snippet: Control lysates tested include those for: HSD17ß1 (human placental whole cell lysate; NB820-59248, Novus Biologicals), HSD17ß2 (human embryonic kidney cells; NBL1-11726, Novus Biologicals), HSD17ß4 (human embryonic kidney cells; NBL1-11728, Novus Biologicals), HSD17ß7 (human embryonic kidney cells; NBP2-07042, Novus Biologicals) and SULTe1 (human embryonic kidney cells; ab94289, Abcam).

Techniques:

Journal: Methods and Protocols

Article Title: Capillary Western Immunoassay Optimization of Estrogen Related Factors in Human Subcutaneous Adipose Tissue

doi: 10.3390/mps5020034

Figure Lengend Snippet: Each electropherogram compares results from overnight acetone precipitation and Minute™ kit preparation of SAT samples. Lysate concentrations of 0.5 and 1.0 mg/mL paired with 1:10 and 1:50 antibody concentration were tested: ( A ) HSD17ß2 ( B ) HSD17ß7 and ( C ) SULTe1.

Article Snippet: Control lysates tested include those for: HSD17ß1 (human placental whole cell lysate; NB820-59248, Novus Biologicals), HSD17ß2 (human embryonic kidney cells; NBL1-11726, Novus Biologicals), HSD17ß4 (human embryonic kidney cells; NBL1-11728, Novus Biologicals), HSD17ß7 (human embryonic kidney cells; NBP2-07042, Novus Biologicals) and SULTe1 (human embryonic kidney cells; ab94289, Abcam).

Techniques: Concentration Assay

Journal: Methods and Protocols

Article Title: Capillary Western Immunoassay Optimization of Estrogen Related Factors in Human Subcutaneous Adipose Tissue

doi: 10.3390/mps5020034

Figure Lengend Snippet: All electropherograms report results from over-expresser lysates at concentrations of 0.5 and 0.8 mg/mL ( A – D ) except for SULTe1 which was tested at: 1/50, 1/100, 1/200, 1/400, 1/800. Empty vector negative controls are also displayed on each electropherogram. Lysates were paired with antibody concentrations of 1:10 and 1:50. Data are listed as follows: ( A ) HSD17ß1, ( B ) HSD17ß2, ( C ) HSD17ß4, ( D ) HSD17ß7, and ( E ) SULTe1.

Article Snippet: Control lysates tested include those for: HSD17ß1 (human placental whole cell lysate; NB820-59248, Novus Biologicals), HSD17ß2 (human embryonic kidney cells; NBL1-11726, Novus Biologicals), HSD17ß4 (human embryonic kidney cells; NBL1-11728, Novus Biologicals), HSD17ß7 (human embryonic kidney cells; NBP2-07042, Novus Biologicals) and SULTe1 (human embryonic kidney cells; ab94289, Abcam).

Techniques: Plasmid Preparation

Journal: Methods and Protocols

Article Title: Capillary Western Immunoassay Optimization of Estrogen Related Factors in Human Subcutaneous Adipose Tissue

doi: 10.3390/mps5020034

Figure Lengend Snippet: Summary of expected and observed molecular weights for all 11 antibodies tested in each experiment.

Article Snippet: Control lysates tested include those for: HSD17ß1 (human placental whole cell lysate; NB820-59248, Novus Biologicals), HSD17ß2 (human embryonic kidney cells; NBL1-11726, Novus Biologicals), HSD17ß4 (human embryonic kidney cells; NBL1-11728, Novus Biologicals), HSD17ß7 (human embryonic kidney cells; NBP2-07042, Novus Biologicals) and SULTe1 (human embryonic kidney cells; ab94289, Abcam).

Techniques: Extraction, Positive Control

Journal: Cancer Science

Article Title: Cancer‐associated fibroblast and M 2 macrophage markers together predict outcome in colorectal cancer patients

doi: 10.1111/cas.12096

Figure Lengend Snippet: mRNA quantitative expression versus immunohistochemistry. (A) DCSIGN, CD163 M2, α‐smooth muscle actin (α‐SMA), fibroblast‐specific protein 1 (FSP1) and fibroblast activation protein (FAP) immunohistochemistry. Representative pictures of high and low protein expression levels of DCSIGN, CD163 M2 macrophage and α‐SMA, FSP1 and FAP cancer‐associated fibroblast (CAF) markers in human tumor samples. Arrows indicate positive cells. Original magnification, ×20. (B) Association between protein and mRNA expression levels. Statistical association between protein and mRNA expression levels of FSP1 and FAP, CAF markers. In addition, a clear trend toward statistical association is observed between protein and mRNA expression levels of DCSIGN and CD163, M2 macrophage markers and the α‐SMA, CAF marker. Bar charts show the average (±confidence interval) of mRNA expression levels of each gene in the low and high protein expression groups.

Article Snippet: Immunohistochemistry analysis and antibodies Anti‐DC‐SIGN antibody [5D7] (Abcam, Cambridge, UK), anti‐human CD163 (clone 10D6; Novocastra, Barcelona, Spain), anti‐alpha smooth muscle actin antibody [1A4] (Abcam), anti‐human S100A4 (DAKO, Glostrup, Denmark), peptide‐affinity purified polyclonal antibody to fibroblast activation protein (FAP) alpha (Imgenex, San Diego, CA, USA) were used for cell immunostaining.

Techniques: Expressing, Immunohistochemistry, Activation Assay, Marker

Journal: Cancer Science

Article Title: Cancer‐associated fibroblast and M 2 macrophage markers together predict outcome in colorectal cancer patients

doi: 10.1111/cas.12096

Figure Lengend Snippet: Kaplan–Meier curves between expression levels of cancer‐associated fibroblast (CAF) and M2 macrophage markers (individually) and disease‐free survival (DFS) in colorectal cancer patients. Association between CD163, a M2 macrophage marker (A), and α‐smooth muscle actin (α‐SMA) (B), fibroblast‐specific protein 1 (FSP1) (C) and fibroblast activation protein (FAP) (D) CAF markers with DFS. “Low expression” refers to a low‐expression tertile for α‐SMA and low‐ and medium‐expression tertiles for CD163, FSP1 and FAP.

Article Snippet: Immunohistochemistry analysis and antibodies Anti‐DC‐SIGN antibody [5D7] (Abcam, Cambridge, UK), anti‐human CD163 (clone 10D6; Novocastra, Barcelona, Spain), anti‐alpha smooth muscle actin antibody [1A4] (Abcam), anti‐human S100A4 (DAKO, Glostrup, Denmark), peptide‐affinity purified polyclonal antibody to fibroblast activation protein (FAP) alpha (Imgenex, San Diego, CA, USA) were used for cell immunostaining.

Techniques: Expressing, Marker, Activation Assay

Journal: Cancer Science

Article Title: Cancer‐associated fibroblast and M 2 macrophage markers together predict outcome in colorectal cancer patients

doi: 10.1111/cas.12096

Figure Lengend Snippet: Kaplan–Meier curves between expression levels of cancer‐associated fibroblast (CAF) and M2 macrophage markers (individually) and overall survival in colorectal cancer patients. Association of CD163, a M2 macrophage marker (A), and fibroblast‐specific protein 1 (FSP1) (C), a CAF marker, with overall survival. α‐Smooth muscle actin (α‐SMA) (B) and fibroblast activation protein (FAP) (D) expression levels, CAF markers, showed a trend towards an association with overall survival.

Article Snippet: Immunohistochemistry analysis and antibodies Anti‐DC‐SIGN antibody [5D7] (Abcam, Cambridge, UK), anti‐human CD163 (clone 10D6; Novocastra, Barcelona, Spain), anti‐alpha smooth muscle actin antibody [1A4] (Abcam), anti‐human S100A4 (DAKO, Glostrup, Denmark), peptide‐affinity purified polyclonal antibody to fibroblast activation protein (FAP) alpha (Imgenex, San Diego, CA, USA) were used for cell immunostaining.

Techniques: Expressing, Marker, Activation Assay

Journal: bioRxiv

Article Title: Tandem Requirement for Full Renal D 1 R and D 5 R Activity

doi: 10.1101/736611

Figure Lengend Snippet: A: In the human renal cortex, D 1 R immunostaining was found throughout the cytoplasm, luminal, and basolateral membranes in renal proximal tubules (PT) and collecting tubule (CT). By contrast, C: D5R immunostaining was noted in luminal but not in basolateral membranes of renal proximal tubules (RPT) or collecting tubule (CT). E: D5R immunostaining was also noted in the medullary thick ascending limbs of Henle (red arrows) but not in medullary collecting ducts (blue arrows) or capillaries (green arrows). No staining was evident in slices with the D 1 R ( B ) and D 5 R ( D, F ) antibody preadsorbed with their respective immunizing peptides. D5R immunostaining was also noted in some of the cultured human renal proximal tubule cells (hRPTCs) (black arrows) ( G ). No staining was evident in cultured hRPTCs with the D 5 R antibody pre-adsorbed with the D 5 R immunizing peptide ( H ). Total cell lysates from hRPTCs treated with vehicle (0) or fenoldopam (1 μM/15 min) were immunoprecipitated (IP) with anti-D 1 R or anti-D 5 R antibodies and immunoblotted for D 5 R or D 1 R, respectively ( I, J ). Normal rabbit (Rb) IgG was used as immunoprecipitant for the negative control. A D 5 R-enriched HEK-293 cell lysate (Novus Biologicals Cat. #NBL1- 10019) (I) or a hRPTC lysate was used for regular immunoblot (IB). ( J )

Article Snippet: Since the D 1 -like receptors share almost the same anatomical distribution at the proximal tubules, we next determined whether the receptors interact in hRPTCs and observed that these receptors co-immunoprecipitated at the basal state (mostly as dimers) and upon treatment (both as monomers and dimers) with the D 1 -like (D 1 R/D 5 R) receptor agonist fenoldopam in D 5 R-enriched HEK293 cells (Novus Biologicals Cat. #NBL1-10019) and hRPTCs ( ).

Techniques: Immunostaining, Staining, Cell Culture, Immunoprecipitation, Negative Control, Western Blot