Ab76020

Paraffin-embedded brain sections were dewaxed and antigen retrieval was performed as described previously (56 (link)). The following primary antibodies were used for immunolabeling: a chicken polyclonal anti-MAP2 antibody (ab5392; Abcam), a rabbit monoclonal anti-Na,K-ATPase α subunit antibody (ab76020, Abcam), and a mouse monoclonal anti-Na,K-ATPase α1 subunit antibody (a6F; DSHB). Confocal microscopy was performed with Zeiss LSM 780 and Leica TCS SP8 microscopes. For further details, see Supporting information (Text S3).

For cycloheximide (CHX) chase experiments, tsA-201 cells (2.2e + 06 cells per 100 mm dish) were seeded and transfected as described. Cells were treated two days after transfection with 20 μg/ml CHX (C1988, Merck) for 2, 4 and 8 h. Immunoblotting was carried out as described above with the following changes: 15 µg of extracted membrane proteins was loaded and detection of the Na/K-ATPase (1:100 000; #ab76020, Abcam) was preferred instead of alpha-Tubulin. Due to space restrictions, WT samples of one transfection were used on two separate plots for comparison with RG and RL protein levels. Thus, the mean of the resulting WT signal intensities after normalization to loading control over these two blots served as one value for WT samples.

Cytoplasm and membrane fractions were mixed with 4x Laemmli and β-mercaptoethanol. Equal volumes of fractions were directly loaded in a 4-12% pre-cast gradient gel (BioRad, USA) and separated by SDS-Page. Proteins were transferred to a nitrocellulose membrane (BioRad, USA), blocked with 5% non-fat dietary milk (NFDM, Carl Roth, Germany), and washed and incubated with the primary antibodies were either diluted in 5% NFDM or 5% bovine serum albumin (BSA, Serva, USA) overnight at 4°C: hIL-1α (1:500, sc-271618, clone G10, Santa Cruz, USA), mIL-1α (1:1,000, AF-400-SP, R&D, USA), GAPDH (1:5,000, sc-47724, clone 0411, Santa Cruz, USA), NaK ATPase (1:10,000, ab76020, Abcam, United Kingdom). The membrane was incubated with the secondary antibody coupled with horse radish peroxidase diluted in 5% NFDM for 1h on the following day. Classico Western HRP Substrate (Millipore) or SuperSigna West Femto (Thermo Fisher Scientific, USA) were used for development on iBright 1500 (Thermo Fisher Scientific, USA).

Membrane protein-enriched samples from cultured cells were used for loading, as the iodide transporter normally functions within the cell membrane. After a PBS wash, the cells were harvested using an elution buffer (20 mmol/L HEPES, pH 7.6, 20 mmol/L NaCl, 0.5 mmol/L EDTA, 10% glycerol) and scraped into a chilled microtube. The cells were sonicated and centrifuged for 30 min at 10,000g at 4 °C, and the pellet was resuspended using a membrane-solubilizing buffer (elution buffer with 1% triton X-100) and incubated on ice for 30 min. After centrifugation, the supernatant was used as the membrane protein-enriched sample. The sample were separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to poly vinylidene fluoride membrane. These membranes were blocked for 30 min at room temperature with 0.5% skim milk in PBS containing 0.1% (v/v) Tween 20 (PBS-T) and then incubated with appropriately diluted rabbit anti-SLC26A7 (1:1000; BMP084, MBL), rabbit anti-SLC26A4 (1:500; ab98091, Abcam), mouse anti- SLC5A5 (1:1000; ab17795, Abcam), or mouse anti-FLAG antibodies (1:1000; F1840, Sigma-Aldrich) as primary antibodies. A rabbit anti-sodium potassium ATPase antibody (1:10000; ab76020, Abcam) and a mouse anti-β-actin (1:10000; A5441; Sigma-Aldrich) antibody were used as internal controls.

The primary antibodies against Kir4.1 (APC-035) and AQP4 (AQP-004) were from Alomone Lab (Jerusalem, Israel). Dystrophin (ab7164), GFAP (ab53554) and Na⁺-K⁺-ATPase (ab76020) antibodies were from Abcam (Cambridge, UK). Glutamine synthetase (GS, NBP2–43646) and VEGF-A (NB100–664) antibodies were from Novus Biological (Littleton, USA). Potassium indicator (P1267MP) and sodium indicator (S1263) were from Thermo Fisher Scientific (Shanghai, China).