Sc 25287

The renal proteins were prepared using Laemmli buffer and separated by SDS-PAGE and then transferred by PVDF membranes. The membranes were blocked with 1% BSA and incubated sequentially with primary antibodies (1:1000 dilution) and secondary antibodies (1:2000 dilution). β-actin was used as the internal control. Western blotting was carried out using antibodies against AQP-1(sc-25287, Santa Cruz Biotechnology, USA), AQP-4 (sc-20812, Santa Cruz Biotechnology, USA), Renin (sc-22671, Santa Cruz Biotechnology, USA), and β-actin (sc-47778, Santa Cruz Biotechnology, USA).

Immunohistochemistry was performed to validate the protein of interest, transmembrane protein 106B (TMEM106B) (52 (link), 73 (link), 83 (link), 84 (link)). The FFPE sections (8 μm) were deparaffinized and rehydrated in a series of xylenes and ethanol dilutions. A heat-induced antigen retrieval was performed with 10 mM sodium citrate, 0.05% Triton-X 100; pH 6. Blocking with 10% normal donkey serum was followed by a TMEM106B primary antibody (1:100, Sigma HPA058342) and AQP1 (1:100, Santa Cruz sc-25287) overnight at 4°C. Sections were incubated with donkey anti-rabbit Alexa-Fluor 647 and Alexa-Fluor 488 secondary antibodies (1:500, Thermo Fisher Scientific, Invitrogen), counterstained with DAPI (Sigma D9542), and coverslipped.
Whole-slide scanning was performed at × 20 magnification with a Leica Aperio Versa 8 microscope using the same settings for each slide. There were three to four images at × 10 magnification collected for each case (n = 5 control, n = 5 AD, n = 5 epilepsy). Images were analyzed using Fiji ImageJ to compare the average amount of TMEM106B positive area among the groups. The same binary threshold was used for all images to determine the number of TMEM106B positive pixels in each image, which was reported as a percentage of the total image area. A Mann–Whitney U-test was performed for statistical analysis; a p-value of < 0.05 was considered significant.

Cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane. To observe the protein expression simultaneously and to ensure the same experimental conditions instead of membrane duplication, we cut the nitrocellulose membrane after transfer into 2 ~ 3 parts by the size; 25 ~ 48 kDa for AQP1 (28 kDa), AQP3 (31 kDa) and AQP4 (34 kDa), 48 ~ 75 kDa for MMP13 (54 kDa) or α-tubulin (55 kDa), 75 ~ 245 kDa for COL2A1 (120 kDa). The membranes were probed with anti-AQP1, -3, -4 (sc-25287, sc-9885, sc-390488, Santa Cruz Biotechnology, Dallas, TX, USA), anti-HMGB1 (ab79823, Abcam, Cambridge, MA, USA), anti-COL2A1 (clone 6B3, MAB8887, Sigma-Aldrich, St. Louis, MO, USA), anti-MMP13 (ab39012, Abcam), and anti-α-tubulin (ab7291, Abcam) antibodies. Immunoreactive proteins were assessed using an LAS-3000 image analyzer (Fuji Film, Tokyo, Japan). The original blot images are provided in ‘Supplementary Figures'. The proteins were quantified by densitometry using ImageJ software (Ver.1.8.0; https://imagej.nih.gov/ij, National Institutes of Health, Bethesda, MD, USA).