Figure 1

KCC2 is expressed in rodent β-cell lines and pancreatic islets. (A–D) Original ethidium bromide stained gels (inverted) showing KCC2 mRNA expression in the mouse β-cell line MIN6 (A), human (B) and mouse islets (C) and the rat INS1-E β-cell line (D). Shown are PCR products of expected sizes representing KCC2a and KCC2b. As the positive control of RT-PCRs, transcripts of GAPDH were amplified (555 bp). As the negative control, water was used instead of total cDNA. (E) Partial chromatograms obtained from representative DNA sequencing reactions using purified KCC2a-671 and KCC2b-602 PCR products from MIN6 (black asterisks). The DNA sequences obtained are 100% identical to the spliced version of KCC2a and KCC2b, respectively. (F) Representative original 1% agarose gel digital image cropped to include bands of relevant sizes. RT coupled to long-range PCR using total RNA from MIN6 and primer sets KCC2a-3591 or KCC2b-4024 (Supplementary Table 1). The PCR products of 3591 bp (KCC2a) and 4024 bp (KCC2b) were purified from this gel, directly cloned in pCR-Blunt II-TOPO vectors and fully sequenced in both directions. (G) MIN6 and mouse brain KCC2a and KCC2b mRNA quantification (n = 5). Results are expressed relative to MIN6 KCC2a. (H) Cropped digitised immunoblots of the indicated protein extracts (µg) obtained from MIN6, mouse (m) and human (h) islets and mouse brain. (I) Digitized immunoblot cropped to show KCC2 protein expression in purified plasma membranes of COS7 cells and MIN6 β-cells. (J,K) Confocal images of MIN6 β-cells immunolabeled using validated KCC2 antibodies. The square in (J) is shown at higher magnification in K to indicate with white arrows immunoreactive KCC2 towards the edges of the cells. Nuclei have been counterstained with DAPI. Scale bar represents 10 µm.