Journal: Biomedicines

Article Title: Comparative Proteomic Analysis of tPVAT during Ang II Infusion

doi: 10.3390/biomedicines9121820

Figure Lengend Snippet: List of up-regulated proteins during Ang II infusion.

Article Snippet: The primary antibodies against KDM1A (BM4356), ALCAM (A01788-1), MBNL1 (A02309-1), and TSN (A02777) were purchased from Boster Biological Technology (Wuhan, Hubei, China).

Techniques: Ubiquitin Proteomics, Migration, Membrane

Journal: Biomedicines

Article Title: Comparative Proteomic Analysis of tPVAT during Ang II Infusion

doi: 10.3390/biomedicines9121820

Figure Lengend Snippet: Immunohistochemical verification of DEPs. ( A ) Immunohistochemistry staining of up-regulated proteins, such as lysine-specific histone demethylase 1A (KDM1A), serine/threonine-protein kinase N1 (PKN1), C-terminal-binding protein 1 (CtBP1), transmembrane protein 41B (TMEM41B), myeloblastin (PRTN3), CD166 antigen (ALCAM), and GRB10-interacting GYF protein 2 (GIGYF2). Positive staining was indicated by brown coloration, and nuclei were stained with hematoxylin in blue. ( B ) Immunohistochemistry staining of down-regulated proteins, such as CCN family member 1 (CCN1), muscleblind-like protein 1 (MBNL1), H/ACA ribonucleoprotein complex subunit 2 (NHP2), retinol dehydrogenase 10 (RDH10), T-cell immunoglobulin and mucin domain-containing protein 4 (TIMD4), and Translin (TSN). Positive cells are indicated by brown coloration.

Article Snippet: The primary antibodies against KDM1A (BM4356), ALCAM (A01788-1), MBNL1 (A02309-1), and TSN (A02777) were purchased from Boster Biological Technology (Wuhan, Hubei, China).

Techniques: Immunohistochemical staining, Immunohistochemistry, Staining, Binding Assay

Journal: Biomedicines

Article Title: Comparative Proteomic Analysis of tPVAT during Ang II Infusion

doi: 10.3390/biomedicines9121820

Figure Lengend Snippet: DEPs with high connectivity degree in PPI analysis between saline and Ang II infusion group.

Article Snippet: The primary antibodies against KDM1A (BM4356), ALCAM (A01788-1), MBNL1 (A02309-1), and TSN (A02777) were purchased from Boster Biological Technology (Wuhan, Hubei, China).

Techniques: Saline, Ubiquitin Proteomics

Journal: Cell Death & Disease

Article Title: The PAX6-ZEB2 axis promotes metastasis and cisplatin resistance in non-small cell lung cancer through PI3K/AKT signaling

doi: 10.1038/s41419-019-1591-4

Figure Lengend Snippet: a , b Expression of CD44, CD133, and ALCAM in the indicated groups of A549 and SPC-A-1 cells were detected by western blotting and immunofluorescence staining (scale bar, 50μm). c Representative images and quantitative analysis of sphere formation in the indicated groups of A549 cells (scale bar, 500μm). d mRNA levels of the stemness-associated genes, NANOG , SOX2 , and OCT4 , by RT-qPCR in the indicated groups of A549 cells. e Quantitative analysis of colony formation assays in the four groups of cells treated with four concentrations of cisplatin (0, 0.25, 0.5, 1μm). f CCK-8 assays were performed to determine the resistance or sensitivity to cisplatin at four concentrations; e , f : * P < 0.05, ** P < 0.01; two-way ANOVA. g Quantitative analysis of flow cytometry results to detect apoptosis in the four groups of cells. ** P < 0.01, *** P < 0.001; two-tailed Student’s t -test

Article Snippet: Antibodies against PAX6 (12323-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), vimentin (10366-1-AP), FSP-1 (16105-1-AP), GAPDH (66004-1-Ig), CD44 (15675-1-AP), CD133 (18470-1-AP), and ALCAM (21972-1-AP), as well as secondary antibodies were obtained from Proteintech Group, Inc. (Wuhan, China); antibodies against EGFR (201012-3F12), WNT5A (619919), ZEB2 (505705), Ki67 (200296), AKT (200323-5A11), phospho-AKT (Ser473) (310021), PI3K (220742), and phospho-PI3K p85alpha (Tyr607) (340790) were purchased from Zenbio (Chengdu, China).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Two Tailed Test

Journal: Cell Death & Disease

Article Title: The PAX6-ZEB2 axis promotes metastasis and cisplatin resistance in non-small cell lung cancer through PI3K/AKT signaling

doi: 10.1038/s41419-019-1591-4

Figure Lengend Snippet: a Tumor volumes of subcutaneous Ctl or si-PAX6 or Vector or PAX6 overexpression xenografts during 21 days of treatment with PBS or CDDP. b , c Representative tumors isolated from nude mice and average tumor weights in the indicated groups; ** P < 0.01, *** P < 0.001; two-tailed Student’s t -test. d IHC staining of PAX6, EMT-associated genes (E-cadherin, N-cadherin, vimentin, FSP-1), stem cell biomarkers (CD44, CD133, ALCAM), and the proliferation marker Ki67 in subcutaneous tumors of mice injected with Ctl vs. si-PAX6, Vector vs. PAX6 (scale bar, 50μm)

Article Snippet: Antibodies against PAX6 (12323-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), vimentin (10366-1-AP), FSP-1 (16105-1-AP), GAPDH (66004-1-Ig), CD44 (15675-1-AP), CD133 (18470-1-AP), and ALCAM (21972-1-AP), as well as secondary antibodies were obtained from Proteintech Group, Inc. (Wuhan, China); antibodies against EGFR (201012-3F12), WNT5A (619919), ZEB2 (505705), Ki67 (200296), AKT (200323-5A11), phospho-AKT (Ser473) (310021), PI3K (220742), and phospho-PI3K p85alpha (Tyr607) (340790) were purchased from Zenbio (Chengdu, China).

Techniques: Plasmid Preparation, Over Expression, Isolation, Two Tailed Test, Immunohistochemistry, Marker, Injection

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

Article Title: CD318 is a ligand for CD6

doi: 10.1073/pnas.1704008114

Figure Lengend Snippet: CD6 interacts with CD318. (A) HT1080 CD166 KO cells are deficient of CD166. The CD166 KO cells were analyzed for CD166 expression by flow cytometry. Thin line, isotype control; thick line, CD166 KO cells stained with an anti-CD166 mAb; thin dash line, WT HT1080 cells stained with the anti-CD166 mAb. Data are representative of five independent experiments. (B) HT1080 CD166 KO cells express CD318. The CD166 KO cells were analyzed for CD318 expression by flow cytometry using the anti-CD318 mAb. Thin line, isotype control; thick line, CD166 KO cells stained with the anti-CD318 mAb. Data are representative of five independent experiments. (C) CD6 binds to WT and CD166 KO cells. WT and CD166 KO cells were incubated with the same concentrations of human IgG1 (control) or recombinant human CD6-Fc fusion protein (1 µM), followed by detecting the cell surface-bound CD6 using an Alexa488-anti-human IgG Ab. Thin line, isotype control; thick line, CD166 KO cells stained with CD6; thin dashed line, WT cells stained with CD6. Data are representative of six experiments. (D) Binding of CD6 onto CD166 KO cells is competitively inhibited by soluble CD318. CD166 KO cells were incubated with recombinant human CD6-Fc fusion protein (1 µM) in the presence of different concentrations of recombinant soluble CD318 (0, 1, and 3 µM), then level of cell surface-bound CD6 was quantitated by flow cytometric analysis after staining the cells with an Alexa488-anti-human IgG Ab. Thin shaded line: isotype control; thick dash line, no rCD318; thin dash line, 1 µM CD318; thin line, 3 µM rCD318. The numbers in parentheses represent the molar concentrations of either rHCD6 or rCD318. Data are representative of six independent experiments. (E) CD6 immunoprecipitates CD318 from the CD166 KO cell lysates. CD166 KO cell lysates were immunoprecipitated with the same concentrations of either soluble CD6 or human IgG1, then the immunoprecipitates were separated by SDS/PAGE and probed with the anti-CD318 Ab, showing that CD6 selectively pulled down CD318 (arrow). Data are representative of eight independent experiments. (F) Soluble CD318 binds to CHO cells that express CD6 on the surface. Control CHO cells (solid line) and CHO cells expressing human CD6 (dotted line) were incubated with recombinant CD318. After washing, the binding of CD318 on the cell surface was assessed by flow cytometry after staining the cells with the anti-CD318 mAb.

Article Snippet: Cells lacking cell-surface CD166 were selected by fluorescence-activated cell sorting using an antibody against the extracellular domain of CD166 (R&D Systems; MAB656).

Techniques: Expressing, Flow Cytometry, Control, Staining, Incubation, Recombinant, Binding Assay, Immunoprecipitation, SDS Page

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

Article Title: CD318 is a ligand for CD6

doi: 10.1073/pnas.1704008114

Figure Lengend Snippet: CD318 is a potential biomarker for inflammatory arthritis and chemotactic for T cells. (A) CD318 is highly expressed in synovial tissues from RA patients. Synovial tissue sections from RA, OA, and nonrelevant controls (NL) were stained with either mAb 3A11 (mouse anti-human CD318) or the same amount of control IgGs, then slides were examined under a microscope. (B) Levels of total CD318 are elevated in synovial tissues from RA patients. Synovial tissue from patients with RA (n = 13), OA (n = 20), and normal synovial tissues (Ctrl, n = 17) were homogenized, and levels of total CD318 were analyzed by ELISA. (C) Levels of soluble CD318 are significantly higher in synovial fluids from patients with RA (n = 36) or JIA (n = 10) than in those from patients with OA (n = 28). Sr, serum; SF, synovial fluid. (D) Soluble CD318 is chemotactic to T cells. T-cell migration toward various concentrations of CD318 (50, 100, 200, 400, 800, and 1,600 pg/mL) was assessed in Boyden chemotaxis chambers. PBS was the negative control, and TARC was the positive control. Readings represent the number of cells migrating through the membrane (the sum of three high power 40x fields per well, averaged for each quadruplicate well). (E) T-cell adhesion to IFN-γ–stimulated synovial fibroblasts in the presence of mouse IgG (control), anti-CD318, anti-CD166, or both was measured by a Synergy plate reader.

Article Snippet: Cells lacking cell-surface CD166 were selected by fluorescence-activated cell sorting using an antibody against the extracellular domain of CD166 (R&D Systems; MAB656).

Techniques: Biomarker Discovery, Staining, Control, Microscopy, Enzyme-linked Immunosorbent Assay, Migration, Chemotaxis Assay, Negative Control, Positive Control, Membrane