Journal:

Article Title: Expression, Localization, and Function of Junctional Adhesion Molecule-C (JAM-C) in Human Retinal Pigment Epithelium

doi: 10.1167/iovs.08-2129

Figure Lengend Snippet: JAM-C expression in the RPE. (A) Quantitative PCR for JAM-A, -B, and -C was performed on RNA extracted from hfRPE cells and RPE cells from adult native tissue. JAM-C mRNA is the most abundant of the three family members in both adult native RPE and cultured hfRPE cells. The data are presented as the mean ± SD of resuslts in three experiments from three donors. (B) Western blot analysis of cultured hfRPE and adult human native RPE with mAb Gi11 specific for human JAM-C. Ezrin was used as a loading control. Similar results were obtained in three experiments. (C) Western blot analysis of cultured hfRPE with two mAbs specific for human JAM-C, mAb Gi11 and mAb PACA-4.

Article Snippet: To confirm the localization of JAM-C in the apical processes of human fetal RPE cells, we used the following three antibodies: (1) Gi11/human epitope (BD Pharmingen, San Diego, CA); (2) AF1213/mouse epitope (R&D Systems, Minneapolis, MN; and (3) PACA4 (generous gift of Tony Liang of Raven Biotechnologies, South San Francisco, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Western Blot

Journal: Cells

Article Title: Pseudomonas aeruginosa Alters Critical Lung Epithelial Cell Functions through Activation of ADAM17

doi: 10.3390/cells11152303

Figure Lengend Snippet: Concentrations and suppliers of used antibodies and important substances. ADAM, a disintegrin and metalloproteinase; CCL2, chemokine (C-C motif) ligand 2; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; JAM-A, junctional adhesion molecule A; TAPI-1, TNF-alpha protease inhibitor I; TRITC, tetramethylrhodamine; WC, working concentration.

Article Snippet: Rabbit anti-human GAPDH was purchased from Santa Cruz Biotech (Dallas, TX, USA), mouse anti-human JAM-A and mouse anti-human Flotilin-1 from BD Biosciences (Heidelberg, Germany), and peroxidase-conjugated anti-mouse and anti-rabbit IgG secondary antibodies from GE Healthcare (Chicago, IL, USA).

Techniques: Protease Inhibitor, Concentration Assay

Journal: Cells

Article Title: Pseudomonas aeruginosa Alters Critical Lung Epithelial Cell Functions through Activation of ADAM17

doi: 10.3390/cells11152303

Figure Lengend Snippet: Impact of ADAM17 on epithelial regeneration. shRNA sequences against ADAM17 (A17KD1 or A17KD2), delivered by lentiviral particles, were used to mediate ADAM17 deprivation in A549 cells while an unspecific shRNA sequence served as scramble control (scr). Cells were grown to confluence and preincubated with either DMSO (0.1%) or TAPI-1 (10 µM) for 30 min. ( A , B ) Cells were treated with mitomycin (5 µg/mL) for 2 h to avoid cell proliferation and then either challenged for 4 h with ExoA (100 ng/mL) or left untreated. Subsequently, the stimulant was removed, an automated scratch was performed and the wound closure was monitored for 24 h using a live cell imaging system. The percentage of wound closure was calculated relative to a fully closed wound. Example images are shown in ( A ). ( C , D ) Cells were challenged with P. aeruginosa for 4 h or left untreated. After 4 h, the cells were lysed and the expression of JAM-A was analyzed by Western blot using an antibody against the N-terminal domain (extracellular part). GAPDH served as a loading control. An exemplary Western blot is shown in ( C ). Quantitative data are shown as mean + SD (n = 7 in ( B ), n = 5 in ( D )). Asterisks indicate significance among treated cells calculated using one-way ANOVA and Tukey post-test ( A , B ) or two-tailed two samples t -test for ( C , D ) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Rabbit anti-human GAPDH was purchased from Santa Cruz Biotech (Dallas, TX, USA), mouse anti-human JAM-A and mouse anti-human Flotilin-1 from BD Biosciences (Heidelberg, Germany), and peroxidase-conjugated anti-mouse and anti-rabbit IgG secondary antibodies from GE Healthcare (Chicago, IL, USA).

Techniques: shRNA, Sequencing, Live Cell Imaging, Expressing, Western Blot, Two Tailed Test

Journal: Cells

Article Title: Pseudomonas aeruginosa Alters Critical Lung Epithelial Cell Functions through Activation of ADAM17

doi: 10.3390/cells11152303

Figure Lengend Snippet: Model of ADAM17 activation during P. aeruginosa infection. S. pneumoniae and P. aeruginosa are recognized through TRL2 and TLR4. ADAM17 activation occurs in a cell-associated manner dependent on the secreted toxin repertoire. Amongst those, ExoA induces the cleavage of junctional adhesion molecules such as JAM-A leading to increased permeability and impairment of regeneration ( A ). Shedding of JAM-A and especially of other adhesion molecules (e.g., VCAM-1, ICAM-1), induced through different exoenzymes (e.g., ExoU), may lead to reduced adhesion of monocytes, limiting the transmigration ( B ). These effects are strongly correlated to reduced lung epithelial cell survival, potentially mediated through TNFR1 shedding ( C ).

Article Snippet: Rabbit anti-human GAPDH was purchased from Santa Cruz Biotech (Dallas, TX, USA), mouse anti-human JAM-A and mouse anti-human Flotilin-1 from BD Biosciences (Heidelberg, Germany), and peroxidase-conjugated anti-mouse and anti-rabbit IgG secondary antibodies from GE Healthcare (Chicago, IL, USA).

Techniques: Activation Assay, Infection, Permeability, Transmigration Assay