Journal: PLOS ONE

Article Title: High-dose radiation-induced immunogenic cell death of bladder cancer cells leads to dendritic cell activation

doi: 10.1371/journal.pone.0307024

Figure Lengend Snippet: Primers used in this study.

Article Snippet: After three washes with TBST, the membrane was placed in a solution of TBST containing specific antibodies against rabbit anti-human GAPDH (10494-1-AP; Protein-tech, Rosemont, USA), HMGB1 (D260488; BBI, Shanghai, China), CRT (AB92516; Abcam, Cambridge, UK), and HSP70 (AB181606, Abcam, Cambridge, UK).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans

doi: 10.3390/ijms23031259

Figure Lengend Snippet: Pathogen-specific regulation of a disintegrin and metalloproteinase (ADAM)10 protein expression and surface localization in bacterial infection. A549 cells were grown to confluence and either left unstimulated or infected with Pseudomonas aeruginosa (P. aeruginosa ) (multiplicity of infection of 5 (MOI 5) ( A , B ), infected with Streptococcus pneumoniae ( S. pneumoniae ) (MOI 5) ( C , D ) or stimulated with exotoxin A (ExoA) (100 ng/mL, E , F ). In ( A – E ), samples were taken after an incubation time of 30, 60, 120 or 240 min. In ( F ), samples were probed after 4 h. ( A , C , E ): ADAM10 protein expression and maturation was investigated in cell lysates by Western blot probing with an antibody against the C-terminus (intracellular part). Probing against glyceraldehyde-3-phosphat dehydrogenase (GAPDH) served as loading control. Band intensities of the pro-form (100 kDa) and the mature form (70 kDa) were quantified by densitometry and normalized to the expression of the unstimulated cells (0 h). A representative blot is shown in ( A , right) (antibody specificity detailed in ). ( B , D , E ): ADAM10 surface expression was investigated by surface staining with an N-terminal antibody against ADAM10 (1 µg/mL) and an APC-coupled secondary antibody (5 µg/mL) and subsequent flow cytometric analysis (quantification as mean fluorescence intensity). The values of the adequate isotype control were subtracted followed by normalization to the unstimulated cells. A representative histogram is shown in ( B , left). Quantitative data are shown as means + SD of three independent experiments. Asterisks indicate significance difference to the control calculated using two tailed two samples t-test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Mouse monoclonal antibody against human CD9 (MM2/57); Rabbit polyclonal antibody against human ADAM10 C-terminus was obtained from Invitrogen (Frankfurt, Germany), rabbit polyclonal antibody against human GAPDH from Santa Cruz Biotech (Dallas, TX, USA).

Techniques: Expressing, Infection, Incubation, Western Blot, Control, Staining, Fluorescence, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans

doi: 10.3390/ijms23031259

Figure Lengend Snippet: Pathogen-specific activation of ADAM10 depends on the toxin repertoire and calcium increase. ( A ) A549 cells were grown to confluence and either left unstimulated or infected with heat-inactivated P. aeruginosa (multiplicity of infection of 5 (MOI 5). Samples were taken after an incubation time of 30, 60, 120 or 240 min. ADAM10 protein expression and maturation was investigated in cell lysates by Western blot probing with an antibody against the C-terminus (intracellular part). Probing against GAPDH served as loading control. Band intensities of the pro-form (100 kDa) and the mature form (70 kDa) were quantified by densitometry and normalized to the expression of the unstimulated cells (0 h). A representative blot is shown in ( A ). ( B , C , D ) A549 cells were grown to 70% confluency on poly-L-lysine coated glass coverslips and either left unstimulated (PBS) or stimulated with ExoA (100 ng/mL) for 4 h in Tyrode’s solution ( B ), calcium free Tyrode’s solution ( C , D ). Subsequently, the cells loaded with 5 μM Fura-2 AM for 45 min at 37 °C followed by calcium signaling recording over 1000 s in Tyrode’s solution ( B ), calcium free Tyrode’s solution ( C ) or calcium free Tyrode’s solution followed by addition of 2 mM calcium after 120 s of calcium signaling recording ( D ). Quantitative data are shown as means + SD of three independent experiments. Asterisks indicate significance difference among treated cells at the indicated time point calculated using two-way ANOVA and Bonferroni post-test for F340/380 ratio over time and one-way ANOVA and Tukey post-test for area under the curve (* p < 0.05, ** p < 0.001, *** p < 0.001, **** p < 0.0001, n.s. not significant). In ( A ), significance was analyzed by two tailed two samples t-test. No significant differences were observed.

Article Snippet: Mouse monoclonal antibody against human CD9 (MM2/57); Rabbit polyclonal antibody against human ADAM10 C-terminus was obtained from Invitrogen (Frankfurt, Germany), rabbit polyclonal antibody against human GAPDH from Santa Cruz Biotech (Dallas, TX, USA).

Techniques: Activation Assay, Infection, Incubation, Expressing, Western Blot, Control, Two Tailed Test