Anti nkcc1

MEFs were grown to 80–90% confluency then collected. Cell pellets were weighed and then resuspended in a proportional volume of phosphate buffered saline (PBS). Equivalent volumes of resuspension were always used for each lysis. Cells were lysed using a cell fractionation kit (Cell Signaling Technologies, #9038) with each buffer supplemented with 1 mM PMSF and 1x protease inhibitor cocktail (Cell Signaling Technologies, #5871).
Lysates were separated by SDS-PAGE on 3–8% Tris-acetate gels (BioRad #3450129) for 2.5 hr at 150V, then transferred to PVDF membrane by wet transfer at 50V for 1 hr. Membranes were blocked in either 5% milk or 5% BSA according to the recommendations of the primary antibody manufacturer. Primary antibodies were as follows: anti-NKCC1 (Cell Signaling Technologies #14581), anti-TCF11/NRF1 (Cell Signaling Technologies #8052). Membranes were incubated in primary antibody at 1:1000 in blocking buffer overnight. IRDye secondary antibody (Abcam #216773) was used for infrared detection at 1:10,000 dilution in blocking buffer for 1 hr. Membranes were scanned on an Odyssey CLx (Li-cor) and analyzed with the accompanying software, Image Studio.

Snap frozen hepatic tissue sections were stained with antibodies against GS (mouse, monoclonal; BD Biosciences) or rhesus family B glycoprotein (goat, polyclonal; Abcam) or ornithine aminotransferase (rabbit, polyclonal; Abcam) or GLT1 (rabbit polyclonal, Abcam) followed by staining with anti-rabbit or anti-mouse or anti-goat antibody and DAPI.
Histological analysis of brain tissue was performed as previously described^{9 (link)}. Mice were killed by i.p. injection of pentobarbital and perfused with 20 mL of physiological saline, followed by perfusion with 250 mL of Zamboni’s fixative [4% (wt/vol) paraformaldehyde and 15% (vol/vol) saturated picric acid in 0.1 M PBS, pH 7.2, 4–6 °C]. Tissue, submerged in 20% (wt/vol) sucrose in PBS (24 h at 4 °C) until complete saturation, and finally frozen in precooled 2-methylbutane (Sigma–Aldrich) at −40 °C before being sliced into 50-μm-thick sections on a cryotome (Frigomobil; Leica). Sections were stained for 48 hours with anti-NKCC1 (rabbit, polyclonal; Cell signaling). All antibodies were diluted 1:500 in PBS containing 0.1% saponin (Sigma–Aldrich) and 5% BSA (GE healthcare). Primary antibodies were labeled for 48 hours with fluorochrome-coupled anti-mouse Cy3 or anti-rabbit FITC antibodies (1:500).

Tissues were homogenized in lysis buffer containing (PBS, 1% w/v Triton-X (Sigma-Aldrich) and protease inhibitor, used according to the manufacturer protocol (Sigma-Aldrich). Blots were probed with anti-NKCC1 (1:1000, rabbit, polyclonal; Cell Signaling), HRP-conjugated anti-rabbit antibodies (1:5000, Cell Signaling), HRP-conjugated anti-β-ACTIN (1:2000, Cell Signaling). Densitometric analysis was performed with the Kodak Image Station 4400, using Kodak Molecular Imaging software.