Extended Data Fig. 2 | Traff icking and function of APEX2-conjugated CaV1.2
subunits in heart. a, Exemplar current–voltage relationship of Ca2+ currents
from cardiomyocytes of α1C–APEX2 mice, acquired in the absence (black trace)
and presence (red trace) of nisoldipine. Insets show series of whole-cell CaV1.2
currents recorded from a series of voltage steps between −40 mV and +60 mV
from a holding potential of −50 mV. Scale bars, horizontal 100 ms, vertical
10 pA/pF. Representative of five experiments. b, Time course of changes in
sarcomere length after superfusion of nisoldipine-containing solution.
Representative of seven experiments. c, Percentage sarcomere shortening in
the presence of nisoldipine. Data are mean ± s.e.m. ****P < 0.0001 by unpaired
two-tailed t-test. n = 12 and 7 cardiomyocytes from left to right. NTG,
nontransgenic. d, Immunof luorescence of cardiomyocytes isolated from mice
expressing α1C–APEX2 and β2B–APEX2, exposed to biotin-phenol and H 2 O 2 or no
H 2 O 2. Staining is with anti-V5 and Alexa594-conjugated secondary antibodies
and streptavidin-conjugated Alexa488, and nuclear labelling is with DAPI.
Scale bar, 5 μm. Representative of 13 and 8 cardiomyocytes from 2 and 3 mice
respectively. e, Streptavidin–HRP blot of lysates from isolated ventricular
cardiomyocytes, representative of five similar experiments. f, Exemplar whole-
cell CaV1.2 currents recorded from cardiomyocytes of α1C–APEX2 transgenic
mice. Black trace, 300 nM nisoldipine; blue trace, 200 nM isoproterenol plus
nisoldipine. Representative of nine cells from two biologically independent
mice. g, As in f, except from β2B–APEX2 mice. Black trace, control; blue trace,
200 nM isoproterenol. Representative of seven experiments from two
biologically independent mice. h, i, Anti-phospho-phospholamban
immunoblot of proteins from cardiomyocytes isolated from α1C–APEX2 and
β2B–APEX2 mice. Cardiomyocytes were exposed to either vehicle or 1 μM
isoproterenol after incubation with biotin–phenol. Blots are representative of
three independent experiments from at least five biologically independent
mice for each genotype. j, As in h and i, except that cardiomyocytes were
isolated from non-transgenic mice without incubation with biotin–phenol.
Blot is representative of three independent experiments from three
biologically independent mice. k, As h and i, except that whole heart was
exposed to 1 μM isoproterenol for 5 min after infusion of biotin-phenol. This
blot is representative of at least five biologically independent mice for no
isoproterenol and at least five biologically independent mice for isoproterenol.
For source gel data, see Supplementary Fig. 1.