|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
|
Fig. 4. Phosphorylation of Cos2 on Ser572 strongly reduces its interaction with
Smo and Ci. (A) Cos2-WT-Myc, Cos2-572A-Myc and Cos2-572D-Myc were
revealed with specific antibodies in the initial cl8 cell lysate before
immunoprecipitation (IP). Notice that endogenous Smo, Cos2 and Fu proteins
were shifted and Ci was stabilised upon Hh treatment. Notice also the presence
of a phospho-Ser signal on endogenous Cos2 and on Cos2-WT-Myc in Hh-expressing
cells. (B) In Cos2 IP, Fu associated similarly with the three different
Cos2 variants. The presence of Smo and Ci was strongly reduced in
Cos2-572D-Myc IP (lanes 7 and 8) compared with Cos2-WT-Myc and Cos2-572A-Myc
IP (lanes 3-6). Two western blots performed from two independent IP
experiments are presented for Smo. Notice also that a portion of Ci was
dissociated from the protein complex in Hh-treated cells expressing Cos2-WT.
This dissociation was less apparent in Cos2-572A-Myc or Cos2-572D-Myc
immunoprecipitates. (C,D) The graphs represent the amount of Smo
(C) or Ci (D) associated with the different Cos2 variants after IP, expressed
as a percentage relative to the amount of Smo (C) or Ci (D) associated with
Cos2-WT in non-Hh-treated cells. The mean of three different experiments is
shown. (E) Structure of the wild-type Cos2 protein and the relative
localisation of the epitopes recognised by the three different Cos2 antibodies
used in the IP experiments. (F,G) Left panels: initial lysates
of cl8 cells analysed with specific antibodies. (F) The same extracts were
immunoprecipitated with an anti-Cos2 or anti-S572P antibody recognising,
respectively, the epitopes shown in blue and in red in E. Under similar
conditions, lysates (G) were also submitted to IP with anti-Fu, anti-Smo and
three different anti-Cos2 antibodies. Notice the strong decrease in Smo and Ci
levels in the IP performed with the S572P antibody. Notice also that
phosphorylated Cos2 is absent following IP performed with the anti-Smo
antibody, but is present following IP performed with the anti-Fu antibody.
|
