|
| Status |
Public on Dec 20, 2022 |
| Title |
T cells, 15min, hBD2, 1 |
| Sample type |
protein |
| |
|
| Source name |
T cells, hBD-2, 15 min stim
|
| Organism |
Mus musculus |
| Characteristics |
cell type: splenic CD4/CD8 T cells
tissue: spleen
strain: C57BL/6
genotype: wildtype
gender: female
treatment: hBD-2
pamchip number: 640079212
|
| Treatment protocol |
PBS or hBD-2 (60µL/mL) were added for 20 min before T cells were stimulated with anti-CD3/anti-CD28 microbeads in a 1:1 bead-to-cell ratio for 15 min.
|
| Growth protocol |
Spleens were isolated from 2 wildtype (WT) C57BL/6 mice and meshed through a 100 µM cell strainer into a PBS filled dish. T cells were isolated using the Pan T cell isolation kit (Miltenyi). 1Mio CD4/CD8 T cells were cultured in 6-well format in 1mlµL RPMI+10%FCS per well.
|
| Extracted molecule |
protein |
| Extraction protocol |
PamGene lysates were prepared according to manufacturers instructions with M-PER Mammalian Protein Extraction Reagent (ThermoFisher) containing Halt phosphatase and Halt protease inhibitor cocktails (ThermoFisher). Total protein was quantified using the Pierce BCA Protein Assay Kit (ThermoFisher).
|
| Label |
PY20 FITC
|
| Label protocol |
We used the PTK PamGene Array according to the manufacturer's instructions
|
| |
|
| Hybridization protocol |
5 μg of protein per well was loaded onto the PamChip and the assay was run on a PamStation96 (PamGene International).
|
| Scan protocol |
The degree of phosphorylation was measured in real time via the kinetic image capture program, Evolve (PamGene International), which captures FITC-labeled anti-phosphotyrosine antibody binding to each phosphorylated peptide substrate. Following 60 min incubation, the signal intensities for each peptide were analyzed using BioNavigator Software (PamGene International).
|
| Description |
Protein lysates with still active kinases, phosphorylation of spotted peptides on chip detected with FITC labeled anti-phospho antibody
|
| Data processing |
Only peptides that showed kinetics (increase of signal in time) on at least 25 % of the arrays were included in the analysis. Nominal coefficient of variation (CV) was calculated per peptide using a 2-component error fit model using overall mean as input and was used as a filter to remove low-intensity spots. Only peptides that showed nominal CV <0.5 were included in the analysis. For kinase interpretation, a ranked list of putative kinases responsible for differences in the peptide phosphorylation was generated by upstream kinase analysis (UKA, PamGene International).
|
| |
|
| Submission date |
Mar 11, 2022 |
| Last update date |
Dec 21, 2022 |
| Contact name |
Natalie Koehler |
| E-mail(s) |
natalie.koehler@uniklinik-freiburg.de
|
| Phone |
+4916099634194
|
| Organization name |
University Medical Center Freiburg
|
| Street address |
Breisacher Str. 115
|
| City |
Freiburg |
| State/province |
NONE |
| ZIP/Postal code |
79106 |
| Country |
Germany |
| |
|
| Platform ID |
GPL32056 |
| Series (2) |
| GSE198404 |
Effect of human beta defensin-2 (hBD-2) on T cells [mouse] |
| GSE198405 |
Effect of human beta defensin-2 (hBD-2) on T cells |
|
