primary antibody mouse anti-cdk7  (Santa Cruz Biotechnology)

Journal: The Journal of investigative dermatology

Article Title: Cyclin-Dependent Kinase 7 Promotes Th17/Th1 Cell Differentiation in Psoriasis by Modulating Glycolytic Metabolism.

doi: 10.1016/j.jid.2021.04.018

Figure Lengend Snippet: Figure 1. CDK7 expression is upregulated in psoriatic CD4D T cells from peripheral blood and lesions. (a) Heat map showing gene expression profiles of differentially expressed proteins in circulating CD4þ T cells from Pso (Psoriasis) compared with age- and sex-matched HC (Healthy) (n ¼ 3 for each group). Colors represent high (red) and low (green) intensity. (b) Representative flow cytometry data showing CDK7 protein expression in circulating CD4þ T cells from Pso and HC. The MFI for CDK7 protein expression was analyzed by flow cytometry (n ¼ 21 for each group, mean SD, two-tailed Student’s t-test, ****P < 0.0001). (c) Correlation of CDK7 protein levels in circulating CD4þ T cells with PASI in Pso (n ¼ 21). The adjusted R2 and P-values are plotted in each graph. P-values were calculated by linear regression. (d) Representative images of immunofluorescence were analyzed for the expression of CD4 (green) and CDK7 (red) in healthy skin and psoriatic lesions. Arrowheads indicate double-positive CD4/CDK7 cells. Nuclei were counterstained with DAPI (blue). Bar ¼ 50 mm (n ¼ 3 for each group). HC, healthy control; MFI, mean fluorescence intensity; HC, healthy control; Pso, patients with psoriasis.

Article Snippet: Cells were stained with PECY7 rat anti-human CD4 antibody (BioLegend, San Diego, CA) for 30 minutes at 4 C, fixed and permeabilized with FOXP3 staining buffer kit (eBioscience, San Diego, CA), and incubated with mouse anti-CDK7 primary antibody (Santa Cruz Biotechnology) for 50 minutes at 4 C. The cells were then washed and stained with secondary FITC anti-mouse IgG antibody (BioLegend) for 30 minutes at 4 C. Gating was performed on single, live, CD4þ T cells.

Techniques: Expressing, Gene Expression, Cytometry, Two Tailed Test, Control

Journal: The Journal of investigative dermatology

Article Title: Cyclin-Dependent Kinase 7 Promotes Th17/Th1 Cell Differentiation in Psoriasis by Modulating Glycolytic Metabolism.

doi: 10.1016/j.jid.2021.04.018

Figure Lengend Snippet: Figure 2. Cdk7 knockdown of CD4D T cells ameliorates psoriasiform symptoms and reduces Th17/Th1 cell differentiation in the IMQ-induced psoriasis-like mouse model. (a) Schematic diagram of mouse experimental protocol. (b) H&E staining of the lesions. Bar ¼ 200 mm. (c) Mean thickness of the epidermis. (d) Representative images of immunofluorescence of IFN-g (green) or IL-17A (green) in the lesions. Bar ¼ 200 mm. (e) qRT-PCR for mRNA expression of Il17a and (f) Ifng in lesional skin. Data are mean SEM. *P < 0.05; ****P < 0.0001. P-values were calculated by one-way ANOVA with Tukey’s post hoc test. n ¼ 5 mice per group. IMQ, imiquimod; NC, normal control; shCDK7, CDK7 short hairpin RNA.

Article Snippet: Cells were stained with PECY7 rat anti-human CD4 antibody (BioLegend, San Diego, CA) for 30 minutes at 4 C, fixed and permeabilized with FOXP3 staining buffer kit (eBioscience, San Diego, CA), and incubated with mouse anti-CDK7 primary antibody (Santa Cruz Biotechnology) for 50 minutes at 4 C. The cells were then washed and stained with secondary FITC anti-mouse IgG antibody (BioLegend) for 30 minutes at 4 C. Gating was performed on single, live, CD4þ T cells.

Techniques: Knockdown, Cell Differentiation, Staining, Quantitative RT-PCR, Expressing, Control, shRNA

Journal: The Journal of investigative dermatology

Article Title: Cyclin-Dependent Kinase 7 Promotes Th17/Th1 Cell Differentiation in Psoriasis by Modulating Glycolytic Metabolism.

doi: 10.1016/j.jid.2021.04.018

Figure Lengend Snippet: Figure 3. Inhibiting CDK7 suppresses CD4D T-cell activation and constrains Th17 and Th1 cell polarization in vitro. (a) Relative mRNA expression of CDK7 gene was measured by qRT-PCR in resting human CD4þ T cells and activated CD4þ T cells from healthy controls stimulated by CD3 and CD28 antibodies for 24 hours (n ¼ 3 for each group). (b) CDK7 gene expression was quantified by qRT-PCR in Th17 and Th1 subsets induced by IL-1b and IL- 23 or IL-12 acquired by in vitro differentiation for 5 days (n ¼ 3 for each group). (c) The percentage of CD69þ cells in the CD4þ T-cell population was evaluated by flow cytometry in human naive CD4þ T cells stimulated with different concentrations of THZ1 (0 nM, 2.5 nM, 5 nM, 10 nM) in the presence of CD3/CD28 antibodies for 24 hours (n ¼ 5 for each group). (d) mRNA expression of IL17A and IFNG (n ¼ 4 for each group). (e) The frequency of IL-17Aþ ROR-gtþ Th17 cells and (f) IFN-gþ T-betþ Th1 cells evaluated by flow cytometry (n ¼ 5 for each group). (g) Correlation of the percentage of ROR-gtþ cells and (h) T-betþ cells in the CD4þ T-cell population with CDK7 MFI (n ¼ 13 for each group). Data are mean SEM. *P < 0.05; **P < 0.01; ***P < 0.001. P-values were calculated by one-way ANOVA with Tukey’s post hoc test (a, b, c, d, e, f) or linear regression (g, h). MFI, mean fluorescence intensity; ns, not significant; Th, T helper type.

Article Snippet: Cells were stained with PECY7 rat anti-human CD4 antibody (BioLegend, San Diego, CA) for 30 minutes at 4 C, fixed and permeabilized with FOXP3 staining buffer kit (eBioscience, San Diego, CA), and incubated with mouse anti-CDK7 primary antibody (Santa Cruz Biotechnology) for 50 minutes at 4 C. The cells were then washed and stained with secondary FITC anti-mouse IgG antibody (BioLegend) for 30 minutes at 4 C. Gating was performed on single, live, CD4þ T cells.

Techniques: Activation Assay, In Vitro, Expressing, Quantitative RT-PCR, Gene Expression, Cytometry

Journal: The Journal of investigative dermatology

Article Title: Cyclin-Dependent Kinase 7 Promotes Th17/Th1 Cell Differentiation in Psoriasis by Modulating Glycolytic Metabolism.

doi: 10.1016/j.jid.2021.04.018

Figure Lengend Snippet: Figure 6. IL-23einduced CDK7 activates the Akt/mTOR/HIF1-a signaling pathway to promote glycolytic metabolism in psoriatic CD4D T cells. (a) Human CD4þ T cells were activated for 2 days in the presence of PBS (normal control) or IL-23 (50 nM)/IL-12 (2.5 ng/ml)/IL-1b (50 nM), respectively, and analyzed for CDK7 expression by flow cytometry. Quantification of MFI of CDK7 is shown (n ¼ 4 for each group). (b) Correlation of CDK7 protein levels in circulating CD4þ

Article Snippet: Cells were stained with PECY7 rat anti-human CD4 antibody (BioLegend, San Diego, CA) for 30 minutes at 4 C, fixed and permeabilized with FOXP3 staining buffer kit (eBioscience, San Diego, CA), and incubated with mouse anti-CDK7 primary antibody (Santa Cruz Biotechnology) for 50 minutes at 4 C. The cells were then washed and stained with secondary FITC anti-mouse IgG antibody (BioLegend) for 30 minutes at 4 C. Gating was performed on single, live, CD4þ T cells.

Techniques: Control, Expressing, Cytometry