γδ T cells were sorted from skin draining lymph nodes (CD3+ γδTCR+) or epidermis (CD45+CD3+ γδTCR+) of WT mice on Eudragit control or eRapa for 1 month using a Becton-Dickinson FACSAria II. αβ T cells were CD3+ γδ TCR−. Due to low cell yield, γδ T cells were pooled from 10 mice/group. 1.5×106 cells were lysed in RIPA buffer and 15 μg protein was ran on a gel and transferred to PVDF membranes as described (6 (link)). Blots were incubated overnight at 4° C with 1:1000 diluted phospho- and/or total antibodies against indicated proteins (all Cell Signaling) plus anti-mouse β-actin (Santa Cruz Biotechnology) and detected by enhanced chemiluminescence (Pierce). Band quantification was by Image J software (National Institutes of Health).
Protocol detail
Western Blot Analysis of γδ T Cells
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Actin
Antibodies
Buffer
Cells
Chemiluminescence
Epidermis
Eudragit
Lymph nodes
Membranes
Mouse
Proteins
Pvdf
Ripa
Skin
T cells
Corresponding Organization : Audie L. Murphy Memorial VA Hospital
Other organizations : Central South University, Urology San Antonio
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Variable analysis
independent variables
- Eudragit control
- ERapa (rapamycin)
- Phosphorylation levels of unspecified proteins
- Total protein levels of unspecified proteins
- Wild-type (WT) mice
- Incubation time: 1 month
- Cell type: γδ T cells from skin draining lymph nodes and epidermis
- Cell isolation: Becton-Dickinson FACSAria II
- Cell lysis: RIPA buffer
- Protein quantification: 15 μg
- Gel electrophoresis and Western blotting
- Primary antibodies: Phospho-specific and total antibodies against unspecified proteins, anti-mouse β-actin
- Detection: Enhanced chemiluminescence
- Quantification: ImageJ software
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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