Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: CD4 + T cells secret p19 after activation through TCR ligation and co-stimulatory signal via CD28 enhances it. ( a ) Mouse T cell hybridomas (DO.BW, 2HCa2, 530, 447, KE8, 216, 118, 1D1-E7, 2B4) and lymphoma (DO11.10) were stimulated with plate-bound anti-CD3 (2 μg/ml ) and anti-CD28 (1 μg/ml ) for 48 h, and culture supernatants were collected and analyzed for p19 by ELISA. ( b – d ) 2B4 cells were stimulated with plate-bound anti-CD3 in the presence or absence of anti-CD28 for indicated time points, and total RNA was extracted and analyzed for mRNA expression by semiquantitative RT-PCR ( b ). PEC macrophages prepared from mice 4 days after the injection with thioglycolate were stimulated with LPS (100 ng/ml) and IFN-γ (1000 U/ml) for 24 h and used as a positive control. Forty-eight hours later, culture supernatants were collected, cell lysates were prepared, and both were analyzed for protein expression of p19 or EBI3 by western blotting ( c ). Culture supernatants from HEK293T cells transfected with expression vectors of pCXN2-p19 or pCXN2-EBI3 were used as positive controls. Culture supernatants of the activated 2B4 cells were also analyzed for p40 together with p19 via ELISA. Culture supernatants of LPS-stimulated splenocytes were used as the positive control ( d ). ( e ) Naive CD4 + T cells from WT mice were stimulated with plate-bound anti-CD3 and anti-CD28 for indicated time points, and total RNA was extracted and analyzed for mRNA expression by semiquantitative RT-PCR. Mouse macrophage cell line, J774.1, and PEC macrophages stimulated with LPS and IFN-γ were used as positive controls. ( f , g) Naive CD4 + T cells were stimulated with plate-bound anti-CD3 (0, 0.2, 0.6 and 2 μg/ml) in the presence or absence of anti-CD28 (1 μg/ml) for 48 h, and total RNA was extracted and analyzed for mRNA expression by semiquantitative RT-PCR ( f ). Culture supernatants of similarly activated naive CD4 + T cells with plate-bound anti-CD3 (0, 0.5, 1 and 2 μg/ml) were collected at the indicated time points and analyzed for IL-2 or p19 by ELISA ( g ). Data are representative of more than two independent experiments.

Article Snippet: The resultant culture supernatants were then concentrated by centrifugation using the Centriprep-10 K centrifugal filter with a 10-kDa Ultracel-10 membrane (Merck Millipore) and subjected to immunoprecipitation with anti-CD5L (D-11, Santa Cruz) followed by western blotting with biotin-conjugated anti-p19 (R&D Systems) as described above.

Techniques: Activation Assay, Ligation, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Injection, Positive Control, Western Blot, Transfection

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: CD4 + T cell-specific conditional p19-deficient mice show alleviated EAE with reduced frequency of GM-CSF + CD4 + T cells in the CNS. Control p19 flox/flox mice or CD4 + T-cell-specific conditional 19-deficient (CD4-Cre/p19 flox/flox ) mice were immunized with MOG 35-55 peptide and their clinical scores were monitored with time ( a ). On day 14, spinal cords and brains were harvested, and the CNS was histopathologically analyzed with H&E staining. Representative images are shown ( b ). On day 41, mononuclear cells were isolated from the CNS, and intracellular cytokine staining was performed after restimulation with PMA and ionomycin. Representative dot plots of GM-CSF and IL-17A in CD4 + T cells are shown ( c ). Average frequencies of respective CD4 + T cells were calculated and compared ( d ). Response to the recall antigen MOG 35-55 peptide was examined using spleen cells ( e ) and mononuclear cells infiltrating the CNS ( f ) on day 14. The culture supernatants were analyzed via ELISA for cytokine production as indicated. Data are shown as mean ± SD (n = 3–7) and are representative of two independent experiments. P values were determined using unpaired, two-tailed Student’s t -test. * P < 0.05, ** P < 0.01.

Article Snippet: The resultant culture supernatants were then concentrated by centrifugation using the Centriprep-10 K centrifugal filter with a 10-kDa Ultracel-10 membrane (Merck Millipore) and subjected to immunoprecipitation with anti-CD5L (D-11, Santa Cruz) followed by western blotting with biotin-conjugated anti-p19 (R&D Systems) as described above.

Techniques: Staining, Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: Differentiation into GM-CSF-producing CD4 + T cells is impaired in p19-deficient CD4 + T cells in vitro. Naive CD4 + T cells from WT mice or complete p19-deficient mice (p19 KO) were stimulated with plate-coated anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) for 4 days under various Th-polarizing conditions; Th, Th0, Th1, ThGM, nonpathogenic Th17, and pathogenic Th17. These cells were then restimulated with PMA and ionomycin, and the intracellular cytokine staining was performed. Representative dot plots for GM-CSF, IL-17A, IFN-γ, and IL-10 in CD4 + T cells are shown ( a ), and average frequencies of respective CD4 + T cells were calculated and compared ( b ). Data are shown as mean ± SD (n = 3) and are representative of three independent experiments. P values were determined using unpaired, two-tailed Student’s t -test. * P < 0.05, *** P < 0.001.

Article Snippet: The resultant culture supernatants were then concentrated by centrifugation using the Centriprep-10 K centrifugal filter with a 10-kDa Ultracel-10 membrane (Merck Millipore) and subjected to immunoprecipitation with anti-CD5L (D-11, Santa Cruz) followed by western blotting with biotin-conjugated anti-p19 (R&D Systems) as described above.

Techniques: In Vitro, Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: p19 associates with CD5L to form a putative heterodimer of p19/CD5L. ( a ) HEK293-F cells were transiently cotransfected with p3 × FLAG-CMV-14-p19 and pCMV3-CD5L-c-MYC and cultured for 3 days, and total cell lysates or culture supernatants were immunoprecipitated with anti-FLAG or anti-c-MYC and control antibody followed by western blotting with biotin-conjugated anti-c-MYC or biotin-conjugated anti-p19, respectively. Immunoprecipitated protein was confirmed by western blotting with the antibody used for immunoprecipitation. ( b ) HEK293T cells were transiently transfected with the control vector p3 × FLAG-CMV-14 alone, p3 × FLAG-CMV-14-p19, pCMV3-CD5L-c-MYC, or both and p3 × FLAG-CMV-14-Hyper-p19/CD5L, then cultured for 3 days. The culture supernatants were then subjected to p19/CD5L-specific ELISA in triplicate. Data are shown as the mean ± SD. ( c ) Naive CD4 + T cells were stimulated with plate-coated anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) under pathogenic Th17-polarizing conditions for 3 days. Resultant culture supernatants were collected and concentrated by centrifugation using a centrifugal filter approximately 8 times, and subjected to immunoprecipitation with anti-CD5L followed by western blotting with biotin-conjugated anti-p19 and subsequently anti-CD5L. ( d ) Naive CD4 + T cells were also similarly stimulated with plate-coated anti-CD3 and anti-CD28 under Th, Th0 and pathogenic Th17-polarizing conditions for 3 days. Resultant culture supernatants were collected and concentrated by centrifugation using a centrifugal filter approximately 10 times, followed by p19/CD5L-specific ELISA in triplicate. ( e ) HEK293-F cells were transiently cotransfected with p3 × FLAG-CMV-14-p19 WT or p3 × FLAG-CMV-14-p19 C55S and pCMV3-CD5L-c-MYC and cultured for 3 days. The culture supernatants were immunoprecipitated with anti-c-MYC and control antibody followed by western blotting with biotin-conjugated anti-p19, then anti-c-MYC. Data are shown as the mean ± SD and representative of four ( a ) or two ( b – e ) independent experiments. P values were determined using one-way ANOVA. * P < 0.05.

Article Snippet: The resultant culture supernatants were then concentrated by centrifugation using the Centriprep-10 K centrifugal filter with a 10-kDa Ultracel-10 membrane (Merck Millipore) and subjected to immunoprecipitation with anti-CD5L (D-11, Santa Cruz) followed by western blotting with biotin-conjugated anti-p19 (R&D Systems) as described above.

Techniques: Cell Culture, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Centrifugation

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: CD5L-deficient mice show alleviated EAE with reduced frequency of GM-CSF + CD4 + T cells in the CNS. ( a – d ) WT mice or CD5L-deficient mice were immunized with MOG 35-55 peptide and their clinical scores were monitored with time ( a ). On day 15, spinal cords and brains were harvested, and the CNS was histopathologically analyzed with H&E staining. Representative images are shown ( b ). Mononuclear cells were also isolated from the CNS, and intracellular cytokine staining was performed after restimulation with PMA and ionomycin. Representative dot plots of GM-CSF, and IL-17A in CD4 + T cells are shown ( c ). Average frequencies of respective CD4 + T cells were calculated and compared ( d ). Blood was also taken over time and serum levels of p19/CD5L and CD5L were determined by ELISA ( e , f ). Data are shown as mean ± SD (n = 4–6) and are representative of three independent experiments. P values were determined using unpaired, two-tailed Student’s t -test ( a , d ) or one-way ANOVA ( e , f ). * P < 0.05, ** P < 0.01, *** P < 0.001. NS, not significant.

Article Snippet: The resultant culture supernatants were then concentrated by centrifugation using the Centriprep-10 K centrifugal filter with a 10-kDa Ultracel-10 membrane (Merck Millipore) and subjected to immunoprecipitation with anti-CD5L (D-11, Santa Cruz) followed by western blotting with biotin-conjugated anti-p19 (R&D Systems) as described above.

Techniques: Staining, Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: p19/CD5L but not either alone efficiently induces cell proliferation, STAT5 phosphorylation. ( a , b ) HEK293T cells were transfected with the control vector p3 × FLAG-CMV-14 alone, p3 × FLAG-CMV-14-p19, pCMV3-CD5L-c-MYC, p3 × FLAG-CMV-14-hyper-p19/CD5L, and p3 × FLAG-CMV-13-hyper-p40/CD5L. The cells were also cotransfected with expression vectors of p3 × FLAG-CMV-14-p19 and pCMV3-CD5L-c-MYC, together with those of p3 × FLAG-CMV-14-p19 and p3 × FLAG-CMV-14-p40, which produced IL-23 as the positive control. The total amount of DNA in each transfection sample was adjusted to be kept equal with the empty vector. Three days later, culture supernatants were collected, and 10% or 30% of them were used for stimulation of Ba/F3 cells expressing gp130/IL-12Rβ1/IL-12Rβ2/IL-23Rα. For p19 + p40 (IL-23), only 10% culture supernatant was added; therefore, “ – ” means that no data exist for the 30% lane ( a ). Ba/F3 cells were also stimulated with purified recombinant p19, CD5L, a mixture of p19 and CD5L (all 20 ng/ml), and hyper-p19/CD5L (0.1 – 20 ng/ml) ( b ). Proliferative activity of these cells was determined by measuring 3 H-thymidine incorporated into the DNA. ( c , d ) Naive CD4 + T cells from WT mice were stimulated with plate-coated anti-CD3 (2 μg/ml ) and anti-CD28 (1 μg/ml ) for 3 days under Th conditions, washed, and rested in 10% FBS medium for 6 h. These cells were unrestimulated ( – ) or then restimulated with purified recombinant p19, CD5L, a mixture of p19 and CD5L, hyper-p19/CD5L, IL-27 (positive control for phosphorylation of STAT1 and STAT3, all 20 ng/ml), or IL-2 (positive control for phosphorylation of STAT5, 100 U/ml) for 5, 15 and 60 min, and subjected to western blotting with anti-pY-STATs and subsequently anti-total STATs ( c ). FACS analysis was also performed using anti-pY-STAT5 and its control antibody after stimulation for 20 min ( d ). Data are shown as the mean ± SD in triplicate and are representative of three ( a , c , d ) or two ( b ) independent experiments. P values were determined using one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The resultant culture supernatants were then concentrated by centrifugation using the Centriprep-10 K centrifugal filter with a 10-kDa Ultracel-10 membrane (Merck Millipore) and subjected to immunoprecipitation with anti-CD5L (D-11, Santa Cruz) followed by western blotting with biotin-conjugated anti-p19 (R&D Systems) as described above.

Techniques: Transfection, Plasmid Preparation, Expressing, Produced, Positive Control, Purification, Recombinant, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: p19/CD5L but not either alone induces augmentation of GM-CSF expression. Naive CD4 + T cells from WT mice were stimulated with plate-coated anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) in the presence of recombinant p19, CD5L (all 20 ng/ml), and hyper-p19/CD5L (2–20 ng/ml) under Th conditions for 3 days, and subjected to intracellular staining of GM-CSF and IFN-γ ( a , b ). Representative dot plots were shown ( a ). Culture supernatants were analyzed for GM-CSF by ELISA ( c ). Similarly, naive CD4 + T cells from CD5L-deficient mice were stimulated and subjected to intracellular staining of GM-CSF and IFN-γ ( d , e ). Representative dot plots were shown ( d ). Culture supernatants were analyzed for GM-CSF by ELISA ( f ). Data are shown as mean ± SD in triplicates and are representative of more than two independent experiments. P values were determined using one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The resultant culture supernatants were then concentrated by centrifugation using the Centriprep-10 K centrifugal filter with a 10-kDa Ultracel-10 membrane (Merck Millipore) and subjected to immunoprecipitation with anti-CD5L (D-11, Santa Cruz) followed by western blotting with biotin-conjugated anti-p19 (R&D Systems) as described above.

Techniques: Expressing, Recombinant, Staining, Enzyme-linked Immunosorbent Assay

Journal: Nanomaterials

Article Title: The Precise Detection of HER-2 Expression in Breast Cancer Cell via Au 25 Probes

doi: 10.3390/nano12060923

Figure Lengend Snippet: Fluorescence imaging of BSA-biotin-fitc labeled HER-2 in SK cells by avidin and primary antibody-biotin at different time points.

Article Snippet: Human HER-2 antibody, HER-2 biotinylated antibody, Avidin and trastuzumab were obtained from Abcam.

Techniques: Fluorescence, Imaging, Labeling, Avidin-Biotin Assay

Journal: Nanomaterials

Article Title: The Precise Detection of HER-2 Expression in Breast Cancer Cell via Au 25 Probes

doi: 10.3390/nano12060923

Figure Lengend Snippet: ( a ) Fluorescence imaging of 12 μM BSA-biotin-Au 25 labeled MDA-MB-231 cells at different time points. ( b ) HER-2 protein expressed in MDA-MB-231 cells before and after paclitaxel stimulation ( c ) Fluorescence imaging of BSA-biotin-Au 25 labeled MDA-MB-231 cells before and after paclitaxel stimulation.

Article Snippet: Human HER-2 antibody, HER-2 biotinylated antibody, Avidin and trastuzumab were obtained from Abcam.

Techniques: Fluorescence, Imaging, Labeling

Journal: Nature Immunology

Article Title: The phosphatidylinositol-transfer protein Nir3 promotes PI(4,5)P 2 replenishment in response to TCR signaling during T cell development and survival

doi: 10.1038/s41590-022-01372-2

Figure Lengend Snippet: a , Total thymocytes from 6- to 8-week-old C57BL/6 female mice were divided into DN, DP, CD4SP and CD8SP populations based on expression of CD4 and CD8α (CD8). b , c , Histograms (b) and quantification (c) of PIP 2 in each thymocyte subset are depicted ( n = 4) MFI, mean fluorescence intensity. d , DN cells were divided into four stages (DN1–DN4) based on the expression of CD44 and CD25. e , DN3 cells were subgated into DN3a and DN3b subsets based on intracellular TCRβ (icTCRβ) and surface expression of CD27 assessed by flow cytometry. f , g , Histograms (f) and quantification (g) of PIP 2 in each DN subset were assessed. h , DP thymocytes were subdivided based on amounts of CD5 and TCRβ (DP1 = CD5 lo TCRβ lo , DP2 = CD5 int TCRβ lo , DP3 = CD5 hi TCRβ lo , DP4 = CD5 hi TCRβ int , DP5 = CD5 hi TCRβ hi ). i , j , Histograms (i) and quantification (j) of PIP 2 in each DP subset were assessed and are presented. iso, isotype control. k , Quantification of TCRβ in each DP subset. n = 4. Data are shown as the mean ± s.e.m. and are representative of three independent experiments. P values were determined using an unpaired, one-way analysis of variance (ANOVA) with multiple comparisons.

Article Snippet: For measurements of PIP 2 in thymocytes using immunostaining, biotinylated monoclonal anti-PIP 2 antibody (clone 2C11, Echelon Biosciences) was used as previously described .

Techniques: Expressing, Fluorescence, Flow Cytometry

Journal: Nature Immunology

Article Title: The phosphatidylinositol-transfer protein Nir3 promotes PI(4,5)P 2 replenishment in response to TCR signaling during T cell development and survival

doi: 10.1038/s41590-022-01372-2

Figure Lengend Snippet: PIP 2 levels on DP thymocytes (DP), CD4 (CD4 T) and CD8 (CD8 T) splenic T cells were measured by flow cytometry. N = 4 mice. Data are shown as the mean ± s.e.m. and are representative of two independent experiments.

Article Snippet: For measurements of PIP 2 in thymocytes using immunostaining, biotinylated monoclonal anti-PIP 2 antibody (clone 2C11, Echelon Biosciences) was used as previously described .

Techniques: Flow Cytometry

Journal: Nature Immunology

Article Title: The phosphatidylinositol-transfer protein Nir3 promotes PI(4,5)P 2 replenishment in response to TCR signaling during T cell development and survival

doi: 10.1038/s41590-022-01372-2

Figure Lengend Snippet: a , Thymocytes and mature T cells from WT and Nir3 –/– male mice were stimulated with anti-CD3 for the indicated times. PIP 2 levels were normalized to unstimulated cells (t = 0). n = 4. b , c , OT-II CD4 T cells expressing Tubby-mScarlet were stimulated on coverslips coated with anti-CD3 antibody. Representative images (b) and quantification (c) of Tubby MFI on the spreading membrane are shown. d – i , OT-I CD8 T cells expressing Tubby-mScarlet were stimulated on coverslips coated with full (OVA), partial (T4) or weak (G4) agonist OVA peptide-MHC tetramers. Representative images (d,f,h) and quantification (e,g,i) of Tubby MFI on the spreading membrane were shown. Scale bar=10μm. N > 8 biological independent cells. All data are shown as the mean ± s.e.m. and are representative of at least two independent experiments. P values were determined using a paired, two-tailed Student’s t -test.

Article Snippet: For measurements of PIP 2 in thymocytes using immunostaining, biotinylated monoclonal anti-PIP 2 antibody (clone 2C11, Echelon Biosciences) was used as previously described .

Techniques: Expressing, Two Tailed Test

Journal: Nature Immunology

Article Title: The phosphatidylinositol-transfer protein Nir3 promotes PI(4,5)P 2 replenishment in response to TCR signaling during T cell development and survival

doi: 10.1038/s41590-022-01372-2

Figure Lengend Snippet: Wildtype (WT) and Nir3 –/– CD3 + T cells were stimulated with bead-bound anti-CD3 and anti-CD28 antibodies for 3 days, transfected with scramble (Scr) or Nir2 siRNAs and rested in IL-7 containing media for 2 days. (a) Nir2 protein levels were assessed by immunoblots 2 days post transfection. (b) Cells were stimulated with 10μg/ml anti-CD3 antibodies (clone 2c11). PIP 2 abundances at indicated time points were assessed by flow cytometry. The data are shown as the mean ± s.e.m. N = 3. Data are representative of two independent experiments.

Article Snippet: For measurements of PIP 2 in thymocytes using immunostaining, biotinylated monoclonal anti-PIP 2 antibody (clone 2C11, Echelon Biosciences) was used as previously described .

Techniques: Transfection, Western Blot, Flow Cytometry

Journal: Nature Immunology

Article Title: The phosphatidylinositol-transfer protein Nir3 promotes PI(4,5)P 2 replenishment in response to TCR signaling during T cell development and survival

doi: 10.1038/s41590-022-01372-2

Figure Lengend Snippet: In immature thymocytes, Nir3, IP 3 R2 and STIM2 are predominantly expressed. Upon weak TCR stimulation from self pMHC complex on thymic stromal cells, a small fraction of PIP 2 is cleaved by PLCγ1 which generates limited amounts of PA at the ER-PM junctions. Nir3 is recruited to the junctions and transfers PI to the PM which is important for PIP 2 replenishment. The whole Nir3-IP 3 R2-STIM2 circuit is critical for the sustained TCR signaling upon weak TCR stimuli. In mature T cells. Nir2, IP 3 R3 and STIM1 are major components of the SOCE pathway. The low sensitivity of the Nir2-IP 3 R3-STIM1 contributes to the non-responsiveness of mature T cells to weak TCR stimuli. Upon strong TCR stimulation from foreign peptide-MHC ligands, a large amount of PIP 2 is metabolized which produces increased PA at the ER-PM junctions. Both Nir2 and Nir3 are recruited to the junctions in tandem. Nir2 transfers PI more efficiently than Nir3. Nir2 is the main PI transfer protein and Nir3 is less required for the PIP 2 replenishment in this context.

Article Snippet: For measurements of PIP 2 in thymocytes using immunostaining, biotinylated monoclonal anti-PIP 2 antibody (clone 2C11, Echelon Biosciences) was used as previously described .

Techniques:

Journal: Cells

Article Title: Protective Effects of SIRT6 Overexpression against DSS-Induced Colitis in Mice

doi: 10.3390/cells9061513

Figure Lengend Snippet: SIRT6 regulated expression and phosphorylation of TAK1. ( a ) The expression and phosphorylation levels of TAK1 were determined by immunohistochemistry. ( b , c ) The statistical results of ( a ). n = 3–6, ** p < 0.01.

Article Snippet: Sections then were blocked in 5% rat serum and incubated overnight at 4 °C with the diluted biotinylated primary antibody (1:500; rat-anti-mouse; anti-TAK1, #ab109526, Abcam, Cambridge, England, UK; anti-p-TAK1, #4508, Cell Signaling Technology, Danvers, MA, USA; anti-c-Jun, #ab40766, Abcam; anti-p-Jun, #ab32385, Abcam; anti-NF-κB p65 antibody, #ab32536, Abcam; anti-NF-κB p65 (phosphor S536), #ab86299, Abcam; anti-NF-κB p65 (acetyl K310) antibody, #ab19870, Abcam).

Techniques: Expressing, Immunohistochemistry

Journal: Biotechnology and bioengineering

Article Title: Human Trabecular Meshwork Cell Behavior is Influenced by Collagen Scaffold Pore Architecture and Glycosaminoglycan Composition

doi: 10.1002/bit.27477

Figure Lengend Snippet: The fibronectin expression quantified on collagen‐only scaffolds using qPCR. Results are shown as a relative fold change by the ΔΔCt method. (*p < .05 and ***p < .005). AFF, aligned flash frozen; NAFF, nonaligned flash frozen; qPCR, qualitative polymerase chain reaction.

Article Snippet: Anti-Fibronectin antibody (Biotin) (ab6584) was obtained from Abcam (Cambridge, UK).

Techniques: Expressing, Polymerase Chain Reaction

Journal: Biotechnology and bioengineering

Article Title: Human Trabecular Meshwork Cell Behavior is Influenced by Collagen Scaffold Pore Architecture and Glycosaminoglycan Composition

doi: 10.1002/bit.27477

Figure Lengend Snippet: Confocal micrographs of collagen‐only scaffolds after week 2 of culture. Fibronectin (red) and nuclei (blue). AFF, aligned flash frozen; CO, collagen only; NAFF, nonaligned flash frozen.

Article Snippet: Anti-Fibronectin antibody (Biotin) (ab6584) was obtained from Abcam (Cambridge, UK).

Techniques:

Journal: Biotechnology and bioengineering

Article Title: Human Trabecular Meshwork Cell Behavior is Influenced by Collagen Scaffold Pore Architecture and Glycosaminoglycan Composition

doi: 10.1002/bit.27477

Figure Lengend Snippet: The fibronectin expression quantified by qPCR on all scaffolds and grouped by the scaffold type. All data are normalized to NAFF–CO at their respective timepoints. (*p < .05). CO, collagen only; CSHA, chondriotin sulfate hyaluronic acid; qPCR, quantitative polymerase chain reaction.

Article Snippet: Anti-Fibronectin antibody (Biotin) (ab6584) was obtained from Abcam (Cambridge, UK).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Biotechnology and bioengineering

Article Title: Human Trabecular Meshwork Cell Behavior is Influenced by Collagen Scaffold Pore Architecture and Glycosaminoglycan Composition

doi: 10.1002/bit.27477

Figure Lengend Snippet: Confocal micrographs of CSHA scaffolds after 2 weeks of culture. Fibronectin (red) and nuclei (blue). AFF, aligned flash frozen; CSHA, chondriotin sulfate hyaluronic acid; NAFF, nonaligned flash frozen.

Article Snippet: Anti-Fibronectin antibody (Biotin) (ab6584) was obtained from Abcam (Cambridge, UK).

Techniques: