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Figure 1.
Figure 1. Phosphorylation on Thr180 of recombinant p38α^D176A
+ F327L is temperature-dependent. (a) Kinase assay of p38α^wt
and p38α^D176A + F327L purified from cultures grown at
different temperatures. The assay measures the ability of the
enzymes to phosphorylate in vitro the GST-ATF2 protein substrate
using [γ-^32P]ATP. Coomassie staining (upper image) verified
the amount of substrate in each lane. The radiograph (lower
image) reveals the activity. (b) The active p38α variant is
threonine phosphorylated. To elucidate phosphorylation we
preformed Western blot analysis utilizing specific anti-phospho
antibodies (anti-phospho-Thr, anti-phospho-Tyr and
anti-phospho-p38). The small arrows indicate the migration
height of p38α in gel electrophoresis. Anti-p38α antibody was
used to determine the amount of p38 loaded at each lane. Some
minor phosphorylation on tyrosine residues can be seen only on
smaller proteolytic products (marked with asterisks). Yet, these
proteins also interacted with the anti-phospho-p38 antibody.
(c) The p38α^D176A + F327L molecule exhibits
autophosphorylation in vitro. Purified p38α^D176A + F327L that
was also used for crystallization was incubated in a kinase
assay buffer without substrate for increasing time intervals at
30 °C. Coomassie staining (upper image) verified the amount
of enzyme in each lane. The radiograph (lower image) reveals
phosphorylation. (d) Mono-phosphorylated p38α is catalytically
active. We measured kinase activity toward GST-ATF2 of
p38α^Y182F, p38α^T180A and p38α^wt activated or not in vitro
by MKK6. Coomassie staining (upper image) verified the amount
of GST-ATF2 in each lane. The radiograph (lower image)
indicates activity. Figure 1. Phosphorylation on Thr180 of
recombinant p38α^D176A + F327L is temperature-dependent. (a)
Kinase assay of p38α^wt and p38α^D176A + F327L purified from
cultures grown at different temperatures. The assay measures the
ability of the enzymes to phosphorylate in vitro the GST-ATF2
protein substrate using [γ-^32P]ATP. Coomassie staining (upper
image) verified the amount of substrate in each lane. The
radiograph (lower image) reveals the activity. (b) The active
p38α variant is threonine phosphorylated. To elucidate
phosphorylation we preformed Western blot analysis utilizing
specific anti-phospho antibodies (anti-phospho-Thr,
anti-phospho-Tyr and anti-phospho-p38). The small arrows
indicate the migration height of p38α in gel electrophoresis.
Anti-p38α antibody was used to determine the amount of p38
loaded at each lane. Some minor phosphorylation on tyrosine
residues can be seen only on smaller proteolytic products
(marked with asterisks). Yet, these proteins also interacted
with the anti-phospho-p38 antibody. (c) The p38α^D176A + F327L
molecule exhibits autophosphorylation in vitro. Purified
p38α^D176A + F327L that was also used for crystallization was
incubated in a kinase assay buffer without substrate for
increasing time intervals at 30 °C. Coomassie staining
(upper image) verified the amount of enzyme in each lane. The
radiograph (lower image) reveals phosphorylation. (d)
Mono-phosphorylated p38α is catalytically active. We measured
kinase activity toward GST-ATF2 of p38α^Y182F, p38α^T180A and
p38α^wt activated or not in vitro by MKK6. Coomassie staining
(upper image) verified the amount of GST-ATF2 in each lane. The
radiograph (lower image) indicates activity.
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