Semi-quantitative analyses of expression and phosphorylation of cSrc
kinase and MYPT1 proteins in middle cerebral arteries from
Atp1a2+/−G301R and WT mice. Representative WT Western
blot where MYPT 1 and cSrc expression and phosphorylation were
studied (a). Thioredoxin 2 (Trx2) was used as loading control. Total
cSrc kinase expression (normalized to Trx2 band density) was reduced
in cerebral arteries from Atp1a2+/−G301R compared to WT
mice (b; n = 6–9). Averaged results suggest an
increased cSrc phosphorylation (calculated as a ratio between
phosphorylated and total cSrc, and normalised to averaged values for
WT under resting conditions) in arteries from
Atp1a2+/−G301R mice compared to WT after U46619
(10−5 M) stimulation (C; n = 5–11).
Incubation with pNaKtide (2 µM) abolished this difference.
Expression of total MYPT1 (normalized to Trx2 band density) was not
different between the groups (n = 4–12; d,
representative Western blot; e, averaged data). U46619 significantly
increased MYPT1 phosphorylation at Thr850
(n = 4–12; f) (calculated as a ratio of
phosphorylated and total MYPT1, and normalised to averaged values
for WT under resting conditions). This potentiation is stronger in
cerebral arteries from Atp1a2+/−G301R than WT. In the
presence of pNaKtide this potentiation was abolished, and there was
no difference between arteries from Atp1a2+/−G301R and
WT. *, ** and ***, P < 0.05, < 0.01
and < 0.001 (two-way ANOVA).