Journal: Infection and Immunity

Article Title: Gingipains from Porphyromonas gingivalis W83 Induce Cell Adhesion Molecule Cleavage and Apoptosis in Endothelial Cells

doi: 10.1128/iai.73.3.1543-1552.2005

Figure Lengend Snippet: FIG. 4. Cleavage of N-cadherin, VE-cadherin, and integrin 1 by gingipain-active extracts from strain W83. Ten micrograms of protein from BCAEC treated in the presence of 5 mM L-cysteine with W83 extracts (70 units of Rgp activity/ml of medium and 5.3 units of Kgp activity/ml of medium) and TLCK-treated W83 (W83 TLCK) and FLL32 extracts (0.73 units of Rgp activity/ml of medium and 0.49 units of Kgp activity/ml of medium) for the indicated times was separated by SDS-PAGE and immunoblotted with a monoclonal antibody to N-cadherin (A) or a polyclonal antibody to VE-cadherin (B). (C) BCAEC treated with W83 extract (200 g/ml) (230 units of Rgp activity/ml of medium and 19.4 units of Kgp activity/ml of medium), FLL32 extract (0.73 units of Rgp activity/ml of medium and 0.49 units of Kgp activity/ml of medium), and W83 extract pretreated with 10 mM TLCK were harvested after treatment for the indicated times, and 20 g was separated by SDS-PAGE. Membranes were reacted with a monoclonal antibody to integrin 1. Arrows indicate cleavage products. Un, untreated.

Article Snippet: The primary antibodies used in these experiments were N-cadherin clone 32 and integrin 1 clone 18 (BD Biosciences), VE-cadherin clone C-19 (Santa Cruz Biotechnology, Santa Cruz, Calif.), poly(ADP-ribose) polymerase (PARP) clone C2-10 (R & D Systems, Minneapolis, Minn.), and a highly specific human autoantibody to topoisomerase I (Topo I) (a generous gift from Eng M. Tan, The Scripps Research Institute, La Jolla, Calif.).

Techniques: Activity Assay, SDS Page

Journal: Infection and Immunity

Article Title: Gingipains from Porphyromonas gingivalis W83 Induce Cell Adhesion Molecule Cleavage and Apoptosis in Endothelial Cells

doi: 10.1128/iai.73.3.1543-1552.2005

Figure Lengend Snippet: FIG. 5. Gingipains can cleave N-cadherin and VE-cadherin immu- noprecipitated from BCAEC. N-cadherin and VE-cadherin were im- munoprecipitated from BCAEC lysates. The immunoprecipitated N- cadherin and VE-cadherin were incubated with 0.125 g of W83 extract (equivalent to 0.19 units of Rgp activity and 0.01 units of Kgp activity), in the presence and absence of TLCK, and 0.125 g of FLL32 extract (0.0037 units of Rgp activity and 0.0025 units of Kgp activity) for the indicated times. Control samples were incubated for the full time course. Samples were separated by SDS-PAGE, and Western blots were performed with antibodies to N-cadherin and VE-cadherin.

Article Snippet: The primary antibodies used in these experiments were N-cadherin clone 32 and integrin 1 clone 18 (BD Biosciences), VE-cadherin clone C-19 (Santa Cruz Biotechnology, Santa Cruz, Calif.), poly(ADP-ribose) polymerase (PARP) clone C2-10 (R & D Systems, Minneapolis, Minn.), and a highly specific human autoantibody to topoisomerase I (Topo I) (a generous gift from Eng M. Tan, The Scripps Research Institute, La Jolla, Calif.).

Techniques: Immunoprecipitation, Incubation, Activity Assay, Control, SDS Page, Western Blot

Journal: bioRxiv

Article Title: Salinomycin disturbs Golgi apparatus function and specifically affects cells in epithelial-to-mesenchymal transition

doi: 10.1101/2022.08.31.506024

Figure Lengend Snippet: (A) Western blot analysis of membrane proteins N- and E-cadherin; epidermal growth factor receptor and transferrin receptor (EGFR and TFRC) in HMLE-shEcad and HMLE-shGFP breast cells (A) or three different breast tumor cell lines (B) that were treated for 24 hours with indicated concentrations of DMSO (Mock), salinomycin (Sal), nigericin (Nig), monensin (Mon) or paclitaxel (PTX). Naphtol blue staining of transferred proteins was used to evaluate equal loading; bloting of E-cadherin in (B) was performed after N-cadherin on the same membrane and it is showing in SUM159 cells, protein bands denoted by #. (C) Western blot analysis of proN-cadherin with specific N-cadherin prodomain antibody in HMLE-Twist cells after treatment with Sal for 24h. (D) Western blot analysis of proN- and N-cadherin in HMLE-Twist cells after treatment with PNGase F. (E) HMLE-Twist and HMLE-pBp cells were treated with DMSO (mock), salinomycin (Sal) or monensin (Mon) for 24 hours and media above cells was subjected to ELISA analysis of secreted IL-8. Data was normalized to mock-treated sample.

Article Snippet: Primary antibodies used: E-cadherin (1:500, Becton-Dickinson, 610181), N-cadherin (1:500, Becton-Dickinson, 610920), proN-cadherin (1:500, R&D Systems, AF1388), EGFR (1:1000, Cell Signaling Technology, 4267), TFRC (1:500, Thermo Fisher Scientific, 13-6800), eIF2α (1:1000, Cell Signaling Technology, 9722), p-eIF2α (1:1000, Cell Signaling Technologies, 3597), CREB-2 (1:100, SCBT, sc-200).

Techniques: Western Blot, Membrane, Staining, Enzyme-linked Immunosorbent Assay