Journal: Scientific Reports

Article Title: Development and application of two novel monoclonal antibodies against overexpressed CD26 and integrin α3 in human pancreatic cancer

doi: 10.1038/s41598-019-57287-w

Figure Lengend Snippet: Immunoprecipitation and immunodetection by Western blot of ( A ) integrin α3 and ( B ) CD26 antigen with novel mAbs KU44.22B and KU44.13A using lysates from CaOV-3 ovarian cancer cells and AsPC-1 pancreatic cancer cells respectively. Left panel: Immunoprecipitation was performed with novel mAbs ( A ) KU44.22B and ( B ) KU44.13A (5 µg) using sheep anti-mouse dynabeads. Protein bands around ~140 KDa and ~ 260KDa were immunoprecipitated with mAb KU44.22B (A; left panel) and ~110 KDa by mAb KU44.13A (B; left panel) respectively and stained with SimplyBlue™ SafeStain. The ~50/25 KDa bands represent heavy and light chains of the anti-mouse antibody. *( B ) left panel corresponds to a cropped gel; vertically sliced images of juxtaposed lanes that were non-adjacent in the gel have a clear separation delineating the boundary between the gels. Middle panel: Integrin α3 and CD26 antigen were immunoprecipitated with mAbs ( A ) KU44.22B and ( B ) KU44.13A (5 µg) respectively, and probed with the same antibody (30 µg/ml). Target antigens were not immunodetected with either of the mAbs. Right panel: Integrin α3 and CD26 antigen were immunoprecipitated with mAbs ( A ) KU44.22B and ( B ) KU44.13A respectively (5 µg) or commercial anti-integrin α3 and anti-CD26 antibodies (2 µg) and immunodetected with commercial mAbs sc-374242 and ab89398 as described in Methods. Immunodetection of target antigens immunoprecipitated by novel mAbs and probed with commercial mAbs confirmed the target identity. MW: molecular weight marker.

Article Snippet: In conclusion, in this study, we reported the production of two novel monoclonal antibodies against integrin α3 and CD26 on human pancreatic cancer cells, using the liver metastasis pancreatic cancer cell line CFPAC-1 as immunogen.

Techniques: Immunoprecipitation, Immunodetection, Western Blot, Staining, Molecular Weight, Marker

Journal: Scientific Reports

Article Title: Development and application of two novel monoclonal antibodies against overexpressed CD26 and integrin α3 in human pancreatic cancer

doi: 10.1038/s41598-019-57287-w

Figure Lengend Snippet: Identification of proteins recognised by novel mAbs KU44.13A and KU44.22B by mass spectrometry.

Article Snippet: In conclusion, in this study, we reported the production of two novel monoclonal antibodies against integrin α3 and CD26 on human pancreatic cancer cells, using the liver metastasis pancreatic cancer cell line CFPAC-1 as immunogen.

Techniques:

Journal:

Article Title: Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

doi: 10.1681/ASN.2007070737

Figure Lengend Snippet: Integrin expression in clone C cells. (A) Expression patterns of integrin β1, αv, α6, and α1 in confluent monolayers of clone C cells seeded at LD and HD. Integrin staining is depicted in red, and nuclear staining with Sytox is shown in green. Integrin α3 staining was also observed by immunostaining but not depicted here. (B) Integrin expression in clone C cells was investigated by basolateral biotinylation followed by immunoprecipitation with integrin antibodies as indicated and Western blotted with anti-streptavidin antibodies (for αv and α3 in the top panel and all lanes in the bottom panel). In addition, Western blots were performed on biotin-streptavidin–purified basolateral membranes from HD cells with integrin α1, α5, and α6. (C) Western blots of cell lysates from LD and HD cells using the polyclonal antibody directed against extracellular domain of rabbit integrin β1 (left). (Right) Western blot using the same antibody in the presence of 600 ng/ml recombinant peptide immunogen. (D) Flow cytometry analysis of clone C cells with various integrin antibodies as indicated in the labels.

Article Snippet: In addition, antibodies against integrin β4 (MAB2058, MAB2060; SC-6628-Santa Cruz Biotechnology, Santa Cruz, CA), integrin β3 (MAB1957; cat. no. 553343-BD Pharmingen, San Jose, CA), integrin α2 (MAB1950), integrin α3 (MAB 1952), and integrin α4 (MAB16983) were also used in immunostaining experiments.

Techniques: Expressing, Staining, Immunostaining, Immunoprecipitation, Western Blot, Purification, Recombinant, Flow Cytometry

Journal:

Article Title: Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

doi: 10.1681/ASN.2007070737

Figure Lengend Snippet: Integrin β1 binds to hensin and integrin αV and gets tyrosine phosphorylated in high density cells. (A) In the first panel, 140 μg of DTSSP–cross-linked cell protein from LD and HD phenotypes were immunoprecipitated with anti–integrin β1 CSAT antibodies, and the blots were probed with guinea pig anti-hensin antibody. In control experiment, membrane containing integrin β1 immunoprecipitates described in the first panel was stripped and reprobed with a different anti–integrin β1 antibody MAB1965 (second panel). Chemically cross-linked cell lysates from LD and HD cells were immunoprecipitated with goat anti-hensin/DMBT1 antibody and blotted with anti–integrin β1 MAB1965 antibody (third panel). Control experiment in which the same samples were probed with guinea pig anti-hensin antibody (fourth panel). (B) Tyrosine phosphorylation of integrin β1. (Left) Anti-phosphotyrosine Western blot of integrin β1 immunoprecipitates (described in A) from LD and HD. (Middle) Control Western blot with integrin β1 antibody. (Right) A streptavidin Western blot of biotinylated basolateral membrane proteins (from HD) that were immunoprecipitated with anti–integrin β1 antibody. (C) Interaction of integrin β1 with integrin α subunits was examined by Western blotting of integrin β1 immunoprecipitates with antibodies to integrin αv, α6, and α1 (left lane in first three panels) and α3 and α5 (last two lanes). Interaction of hensin with integrin α subunits was examined by Western blotting of hensin immunoprecipitates with antibodies to integrin αv, α6, and α1 (right lane in first three panels) from HD cells. (D) Interaction of integrin αv with integrin β subunits was examined by Western blotting of integrin αv immunoprecipitates with integrin β1 (first lane), integrin β3 (second lane), and integrin β5 (third lane) antibodies.

Article Snippet: In addition, antibodies against integrin β4 (MAB2058, MAB2060; SC-6628-Santa Cruz Biotechnology, Santa Cruz, CA), integrin β3 (MAB1957; cat. no. 553343-BD Pharmingen, San Jose, CA), integrin α2 (MAB1950), integrin α3 (MAB 1952), and integrin α4 (MAB16983) were also used in immunostaining experiments.

Techniques: Immunoprecipitation, Western Blot

Journal:

Article Title: Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

doi: 10.1681/ASN.2007070737

Figure Lengend Snippet: Effect of anti-integrin antibodies on cell height and cross-sectional area a

Article Snippet: In addition, antibodies against integrin β4 (MAB2058, MAB2060; SC-6628-Santa Cruz Biotechnology, Santa Cruz, CA), integrin β3 (MAB1957; cat. no. 553343-BD Pharmingen, San Jose, CA), integrin α2 (MAB1950), integrin α3 (MAB 1952), and integrin α4 (MAB16983) were also used in immunostaining experiments.

Techniques:

Journal:

Article Title: Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

doi: 10.1681/ASN.2007070737

Figure Lengend Snippet: Role of integrin αv and β1 in terminal differentiation of clone C epithelia. (A) Clone C cells cultured at high density in media containing integrin αv and β1 function-blocking antibodies or integrin α1 function-blocking antibody. Apical and basal actin stained with phalloidin (red) and imaged by confocal microscopy showing en face and XZ optical sections. (B) HD cells cultured with or without integrin function-blocking antibodies as indicated in the labels were examined for their ability to endocytose fluorescein-dextran. Fluorescein-dextran in green and nuclear stain (Sytox red) in red. (C, top) Clone C cells seeded at low density and allowed to form confluent monolayers, when examined by F-Actin staining, have stress fibers on the basal surface and very little apical actin staining (left); however, when the same experiment is carried out in the presence of integrin β1–activating antibody P4G11, apical actin staining was observed with a marked difference in stress fiber staining on the basal surface. (Bottom) LD cells cultured in the absence or presence of integrin β1–activating antibody P4G11 were examined for their ability to endocytose green fluorescence (FITC) dextran. Nucleus was visualized with Sytox Red dye.

Article Snippet: In addition, antibodies against integrin β4 (MAB2058, MAB2060; SC-6628-Santa Cruz Biotechnology, Santa Cruz, CA), integrin β3 (MAB1957; cat. no. 553343-BD Pharmingen, San Jose, CA), integrin α2 (MAB1950), integrin α3 (MAB 1952), and integrin α4 (MAB16983) were also used in immunostaining experiments.

Techniques: Cell Culture, Blocking Assay, Staining, Confocal Microscopy, Fluorescence

Journal:

Article Title: Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

doi: 10.1681/ASN.2007070737

Figure Lengend Snippet: Integrins αv and β1 mediate ECM deposition of hensin. (A) ECM hensin staining: Clone C cells seeded at high density were cultured with various function-blocking integrin antibodies and exposed to anti-hensin antibodies (followed by rhodamine secondary antibodies) before fixation or permeabilization. Nuclear staining is shown in green. (B) Control experiment to examine intracellular hensin: Clone C cells seeded at high density were cultured with function-blocking integrin antibodies and exposed to anti-hensin antibodies after fixation and permeabilization. (C) Hensin secretion in basolateral medium: Equal amounts of combined basolateral medium from HD cells cultured in the presence and absence of integrin αv–and β1–blocking antibodies collected for 3 d was precleared with agarose beads, immunoprecipitated with anti-hensin (DMBT1) antibodies, and probed with anti-hensin (SRCR6/7) antibodies. (D) Clone C cells seeded at low density and cultured in the absence or presence of activating antibodies to integrin β1 and stained with hensin antibodies before (top) and after (bottom) permeabilization. (E) ECM was extracted from clone C cells seeded at identical low densities but cultured in the absence or presence of integrin β1–activating antibodies for 5 d. The ECM samples were then probed with Western blotting with guinea pig anti-hensin antibody.

Article Snippet: In addition, antibodies against integrin β4 (MAB2058, MAB2060; SC-6628-Santa Cruz Biotechnology, Santa Cruz, CA), integrin β3 (MAB1957; cat. no. 553343-BD Pharmingen, San Jose, CA), integrin α2 (MAB1950), integrin α3 (MAB 1952), and integrin α4 (MAB16983) were also used in immunostaining experiments.

Techniques: Staining, Cell Culture, Blocking Assay, Immunoprecipitation, Western Blot

Journal:

Article Title: Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

doi: 10.1681/ASN.2007070737

Figure Lengend Snippet: Integrin α6 is a candidate for transmitting signals from ECM hensin to the cell interior. Effect of function-blocking α6 integrin antibodies on clone c cells seeded at low density on preformed hensin matrix and cultured for 5 d. Monolayers were then stained with Phalloidin (red) and Sytox (green), and the combined projection of all confocal sections is presented in these panels. XZ section represents a typical cross-section through the monolayer.

Article Snippet: In addition, antibodies against integrin β4 (MAB2058, MAB2060; SC-6628-Santa Cruz Biotechnology, Santa Cruz, CA), integrin β3 (MAB1957; cat. no. 553343-BD Pharmingen, San Jose, CA), integrin α2 (MAB1950), integrin α3 (MAB 1952), and integrin α4 (MAB16983) were also used in immunostaining experiments.

Techniques: Blocking Assay, Cell Culture, Staining