Fig 1: PCR, pharmacology and gene silencing is consistent with the expression of the KCa1.1 ß3 subunit in U-87 MG cells. (A) Results of qPCR experiments assessing the relative expression of the ß subunits in the U-87 MG cell line compared to the pore-forming a subunit (black bar). ß1, ß2 and ß3 mRNA levels divided by the expression of the KCa1.1 channel a-subunit are represented with green, blue and purple bars, respectively (N = 3, n = 3). (B) Western blot of the untreated and gene-silenced U-87 MG cells. The populations were tested in duplicate, the left two lanes are the untreated U-87 MG cells and the right two lanes are untreated (non-transfected and not gene silenced) CHO cells. The middle lane pairs are for the KCNMB1 (ß1), KCNMB2 (ß2) and KCNMB3 (ß3) silenced populations of U-87 MG, from top to bottom, respectively. The thick band in the top box at 42 kDa corresponds to actin and the 22 kDa marker to the ß1 subunit (KCNMB1 gene product). In the second box (middle) the ß2 band (27 kDa marker, KCNMB2 gene product), and in the third box (bottom) the ß3 band (KCNMB3 gene product) are shown (31 kDa marker). (C to E) Pharmacological studies after gene silencing (NSilence = 3). Whole-cell currents were recorded as in Fig. 1D, peak currents were measured and normalized peak currents were calculated as in Fig. 2A and B. (C) Effect of 75 µM LCA on control scrambled siRNA (dark green bar, n = 8) and on ß1 silenced (KCNMB1 siRNA transfected, light green bar, n = 8) U-87 MG cells. (D) Effect of 30 µM AA on the ß2 silenced (KCNMB2 siRNA transfected, light blue bar, n = 6), and (E) on the ß3 silenced (KCNMB3 siRNA transfected, light purple bar, n = 6), compared to the scrambled siRNA transfected groups (dark blue and dark purple bars respectively, n = 7). Insets in E) show the raw currents obtained in a scrambled RNA (left) and in a KCNMB3 siRNA treated cell before (Control, black current traces) and after the application of 30 µM AA (purple current traces). Voltage protocols and solutions are as in Fig. 1D. Throughout the figure, bars and error bars indicate mean ± SEM for the indicated number of experiments (black symbols), *p < 0.05.