to a mutant form lacking the PDZBM in order to identify PDZ and
non PDZ-mediated interactions. In our lab, we systematically combine side by side immunopurification and peptide-based purification procedures to identify PDZ proteins interacting with a
PDZBM containing protein, here TANC1.
2 Materials
2.1 Common
Reagents and Buffers
1. HEK293T cells from ATCC
® Number: CRL-3216™.
2. Dulbecco’s modified Eagle’s medium (DMEM), 4.5 g/L glucose, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and
1 mM glutamine) (Thermo Fisher Scientific).
3. PBS.
SNV
1
1861 a.a.
896
1272 1289 1403
Ankyrin repeat
region
TPR repeat
region
PDZ binding motif
FLAG-TANC1
DAPI
DAPI
A)
B)
Fig. 2 Description and expression of TANC1, a PDZBM containing protein. (a) TANC1 protein is composed by
1601 amino acids with eleven ankyrin repeats, three TPR repeats and a PDZ binding motif located at its
C-terminus. (b) Subcellular localization of FLAG-TANC1. HEK293T expressing FLAG-TANC1 (left) or a carrier
plasmid (right) are grown on coverslips pretreated with poly-L-lysine to let the cells to attach. After 48 h, cells
are fixed with paraformaldehyde 4%, permeabilized using Triton X-100 at 0.4% and labeled with anti-FLAG
antibody and DAPI
Identification of Associated PDZ Proteins
19