Proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) on a 6% separating and 4% stacking gel (for
SERCA 2), on a 4% separating and 3% stacking gel (for IP3R and RyR) and on a 10% separating and 4% stacking gel (for calreticulin) and transferred to nitrocellulose membranes (Hybond ECL Membrane, Amersham Biosciences, UK). After blocking for 2 hours in a 5% solution of non-fat dried milk/TBST (TBS with 0.05% Tween 20), the membranes were incubated overnight at 4°C with specific antibodies (SERCA 2 Abcam 1:1000, Mouse anti-Ryanodine Receptor Chemicon 1:500, Mouse anti-IP3 Receptor Chemicon 1:500, anti-Calreticulin antibody Sigma-Aldrich 1:4000, Beta Actin Antibody (HRP) Loading control Abcam 1:5000, SERCA1 ATPase antibody [VE121G9] Abcam 1:500, SERCA3 ATPase antibody Abcam 1:200). I-BET-762 mw Sheep anti-mouse IgG horseradish peroxidase linked whole antibodies (Amersham Biosciences, UK, 1:1500) were used as secondary antibodies. β-actin served as
a loading control. Antibody complexes were visualized using Hyperfilm ECL chemiluminescence (Amersham Biosciences, UK) and evaluated using the “”Image-J”" analysis PU-H71 supplier software. Statistics One-way ANOVA or “”ANOVA repeated measurements”" (combined with pairwise multiple comparisons) were performed using the “”Sigma Stat”" software (Jandel Scientific, Chicago, IL). A P value of less than 0.05 was considered statistically significant. Results To investigate the role of Ca2+-influx in Ca2+-homeostasis in lung cancer cells, NHBE (normal human bronchial epithelial), H1339 (small cell lung carcinoma), HCC (adeno carcinoma), EPLC 272 (squamous cell carcinoma) and LCLC (large cell lung carcinoma) cells were exposed to 1 mM ATP in the presence and the absence of extracellular calcium (PBS containing no calcium but 0.02% EGTA). The resulting increase in the [Ca2+]c was AZD9291 concentration quantified using fluorescence microscopy. Baseline fluorescence values were similar in all cell lines
Carnitine dehydrogenase (data not shown). In NHBE, H1339 and HCC cells, the ATP-induced Ca2+-increase was comparable with and without external calcium suggesting an insignificant role for Ca2+-influx (Figure 2). In EPLC 272 and LCLC cells, the ATP-induced Ca2+-increase was lower in the absence of extracellular calcium. Figure 2 Cells were exposed to 1 mM ATP in the presence and the absence of extracellular calcium. The resulting increase in the cytoplasmic Ca2+-concentration was quantified using fluorescence microscopy. For each cell line, the Ca2+-increase with external calcium was set to 100% (black columns) and the Ca2+-increase without external calcium (white columns) was expressed as percent of the increase with external calcium. In normal bronchial epithelial (NHBE), Small Cell Lung Cancer (H1339) and Adeno-Carcinoma (HCC) cells, the ATP-induced Ca2+-increase was independent of the presence of extracellular calcium suggesting a minor role for Ca2+-influx.