3.5.2 Validation
of the Newly Identified
TANC1-Associated PDZ
Proteins
Among the proteins associated to TANC1 by immunoprecipitation
and peptide-based purification, we found SCRIB (Figs. 3b and 4b),
a scaffold protein which regulates epithelial and neuronal functions
[13]. We first confirmed by co-immunoprecipitation followed by
western blot analysis that endogenous SCRIB is associated to
TANC1 through its PDZBM (Fig. 5a). To do so, we transfected
HEK293T cells with plasmids encoding either full length FLAGTANC1 or its mutant version (FLAG-TANC1-AAA). We observed
that endogenous SCRIB co-immunoprecipitate with full length
TANC1 but not with the mutant form. These results validate our
MS analysis and confirm that the SCRIB-TANC1 association relies
on the PDZBM of TANC1. Furthermore, to confirm that this
interaction exists at the endogenous level, we immunoprecipitated
endogenous TANC1 using a specific antibody and checked for the
presence of SCRIB (Fig. 5b). We confirmed by western blot analysis
that SCRIB is associated with TANC1 at the endogenous level.
3.6 Discussion
In this chapter, we used immuno- and peptide-based purification
protocols to identify PDZ domain proteins associated to TANC1, a
protein containing a PDZBM. Our results show that the combined
approaches identified a set of 5 PDZ domain proteins which gives
high confidence in the obtained results. The peptide-based
approach was more efficient as it was able to identify four times
more PDZ domain proteins associated to TANC1. Immunopurification of TANC1 using the FLAG sequence allows the purification
of interactors of an entire and structurally folded protein. Furthermore, the given protein of interest (here TANC1) is expressed in its
natural subcellular localization (cytosol, plasma membrane. . .) and
has access to a limited number of interactors. Peptide-based purification relies on peptide-coated beads which provide more binding
IP: α-FLAG
WB: α-FLAG
Lysate
WB: α-SCRIB
IP: α-FLAG
WB: α-SCRIB
1
2
3
FLAG-Tanc1
FLAG-Tanc1 (-AAA)
+
+
-
-
-
-
IP
α-SCRIB
α-TANC1
L y s a t e
C
t l
T A N
C
1
B)
A)
Fig. 5 Validation of the interaction between TANC1 and SCRIB. (a) HEK293T cells
expressing either wild type TANC1 or its mutant form. Following FLAG immunoprecipitation, presence of SCRIB in immunoprecipitates is validated by western
blot analysis. TANC1 is associated to SCRIB through its last three amino acids.
(b) Validation of the TANC1-SCRIB association at the endogenous level. TANC1 is
immunopurified using specific antibodies (Bethyl) and the presence of SCRIB
(antibody from Santa Cruz Biotechnology) is shown by western blot analysis
Identification of Associated PDZ Proteins
35
of the Newly Identified
TANC1-Associated PDZ
Proteins
Among the proteins associated to TANC1 by immunoprecipitation
and peptide-based purification, we found SCRIB (Figs. 3b and 4b),
a scaffold protein which regulates epithelial and neuronal functions
[13]. We first confirmed by co-immunoprecipitation followed by
western blot analysis that endogenous SCRIB is associated to
TANC1 through its PDZBM (Fig. 5a). To do so, we transfected
HEK293T cells with plasmids encoding either full length FLAGTANC1 or its mutant version (FLAG-TANC1-AAA). We observed
that endogenous SCRIB co-immunoprecipitate with full length
TANC1 but not with the mutant form. These results validate our
MS analysis and confirm that the SCRIB-TANC1 association relies
on the PDZBM of TANC1. Furthermore, to confirm that this
interaction exists at the endogenous level, we immunoprecipitated
endogenous TANC1 using a specific antibody and checked for the
presence of SCRIB (Fig. 5b). We confirmed by western blot analysis
that SCRIB is associated with TANC1 at the endogenous level.
3.6 Discussion
In this chapter, we used immuno- and peptide-based purification
protocols to identify PDZ domain proteins associated to TANC1, a
protein containing a PDZBM. Our results show that the combined
approaches identified a set of 5 PDZ domain proteins which gives
high confidence in the obtained results. The peptide-based
approach was more efficient as it was able to identify four times
more PDZ domain proteins associated to TANC1. Immunopurification of TANC1 using the FLAG sequence allows the purification
of interactors of an entire and structurally folded protein. Furthermore, the given protein of interest (here TANC1) is expressed in its
natural subcellular localization (cytosol, plasma membrane. . .) and
has access to a limited number of interactors. Peptide-based purification relies on peptide-coated beads which provide more binding
IP: α-FLAG
WB: α-FLAG
Lysate
WB: α-SCRIB
IP: α-FLAG
WB: α-SCRIB
1
2
3
FLAG-Tanc1
FLAG-Tanc1 (-AAA)
+
+
-
-
-
-
IP
α-SCRIB
α-TANC1
L y s a t e
C
t l
T A N
C
1
B)
A)
Fig. 5 Validation of the interaction between TANC1 and SCRIB. (a) HEK293T cells
expressing either wild type TANC1 or its mutant form. Following FLAG immunoprecipitation, presence of SCRIB in immunoprecipitates is validated by western
blot analysis. TANC1 is associated to SCRIB through its last three amino acids.
(b) Validation of the TANC1-SCRIB association at the endogenous level. TANC1 is
immunopurified using specific antibodies (Bethyl) and the presence of SCRIB
(antibody from Santa Cruz Biotechnology) is shown by western blot analysis
Identification of Associated PDZ Proteins
35