Figure 5.

Knockdown of AKAP18δ affects Ca²⁺ re-uptake in the sarcoplasmic reticulum. Kinetics of Ca²⁺ release and re-uptake in the sarcoplasmic reticulum of depolarized cardiac myocytes transfected with the D1ER sensor alone (red curves), together with control siRNA (blue curves) or AKAP18δ siRNA (green curves) in the presence (open symbols) or absence (filled symbols) of 10 μM norepinephrine (NE; the arrow indicates the time of NE addition). SR Ca²⁺ was depleted by 50 μM BHQ, the cells were washed and extracellular Ca²⁺ was added. siRNA was Cy3-labelled and transfected cells were thus identified. For clarity, only every second data point is shown. Note: timescale differs at breakpoint. The time constant averages (τ, mean±s.e.m.) were calculated excluding outlier values outside mean±2 s.d. (right). For each sample, 10–17 independent cells were analysed (^*P<0.025, by Student's t-test and one-way ANOVA for paired and independent samples, respectively; NS, not significant). Immunoblot (IB, right): siRNA efficacy tested in easily transfectable HaCaT cells expressing GFP–AKAP18δ. Tx. ctr.: transfection control, an unrelated Flag-tagged construct (β-arrestin) was co-transfected and detected by anti-Flag to control for transfection and loading. AKAP, A-kinase anchoring protein; BHQ, 2,5-di-tert-butylhydroquinone; GFP, green fluorescent protein; siRNA, short interfering RNA; SR, sarcoplasmic reticulum.