Fig. 2
Diverse Ca2+ pump expressions were found in breast tumor cell lines representing different subtypes. Basal protein expression of PMCA and SERCA isoforms was analyzed by Western blotting in different breast cancer cell lines. a: Equal amount (30 μg) of total cell protein lysates of confluent cell cultures were immunostained with isoform specific antibodies: anti-pan-PMCA (5F10), anti-PMCA1 (NR1), anti-PMCA2 (NR2), anti-PMCA3 (NR3), anti-PMCA4 (JA9), anti-PMCA4b (JA3), anti-SERCA2 (IID8) and anti-SERCA3 (PL/IM 430). β-actin served as a loading control. Microsomal membrane preparations isolated from COS-7 cells were used as isoform-specific positive controls: PMCA1: 1 μg membrane protein from untransfected cells; PMCA2: a mixed sample of 0.1–0.1 μg membrane protein from cells transfected with rPMCA2a, rPMCA2b, hPMCA2wb constructs; PMCA3: a mixed sample of 0.2–0.2 μg membrane protein from cells transfected with rPMCA3a, rPMCA3b constructs; PMCA4: a mixed sample of 0.2–0.2 μg membrane protein from cells transfected with hPMCA4a, hPMCA4xb constructs. b: Relative protein expression of PMCA1, PMCA4, SERCA2 and SERCA3 isoforms. Densitometric values were normalized to the respective β-actin levels and compared to the protein expression of the non-tumorigenic MCF-10A cells in the case of PMCA1, PMCA4 and SERCA2, or to the protein expression of MCF-7 in the case of SERCA3. Bars represent mean ± SEM from two independent experiments