Fig. 4

PFK15 causes PMCA inhibition in MIA PaCa-2 but not human pancreatic stellate cells: PMCA activity was measured in MIA PaCa-2 cells and HPSCs perfused under constant SERCA blockade (cyclopiazonic acid, CPA, 30 μM) and calcium-free conditions and 1 mM EGTA (black bar). Subsequent ER Ca²⁺ depletion caused store-operated calcium entry when 20 mM Ca²⁺ was introduced (white bar) calcium efflux was used as a measure of PMCA activity before and after 30 min incubation with or without 10 μM PFK15. Representative traces showing clearance experiments with PFK15 in MIA PaCa-2 (a) and HPSCs (b). The rate of clearance was calculated for the fastest 2 min of the first clearance phase (R₁), the clearance rate from the same starting ratio was then calculated in the second clearance phase (R₂). R₁/R₂ was calculated for both MIA PaCa-2 and HPSC with and without PFK15 treatment (c). Membrane and cytosolic protein samples from MIA PaCa-2 cells were produced using a membrane protein extraction kit and PFKFB3 levels assessed by immunoblot. PMCA4 was used as a positive control for membrane samples and GAPDH was used as a positive control for cytoplasmic samples, a representative image is shown (d). Bars represent the mean ± SEM of 3–5 experiments. Kruskall-Wallis test; **p < 0.005