D4c6r

Samples were lysed in western blot buffer [4% (w/v) SDS, 20% (v/v) glycerol and 0.01% bromophenol blue, 0.125 M Tris-hydrochloride, pH 6.8] with 100 mM dithiothreitol (DTT, Thermo Fisher Scientific) as described (Rai et al., 2021c (link)). Membranes were incubated with primary mouse or rabbit antibodies against ALIX (1:1,000 dilution) (3A9; Cell Signaling Technology), TSG101 (1:1,000 dilution) (622696; BD Biosciences), GAPDH (1:1,000 dilution) (D4C6R; Cell Signaling Technology), estrogen receptor alpha (ERα) (1:500 dilution) (D6R2W; Cell Signaling Technology), progesterone receptor A/B (PR A/B) (1:500 dilution) (D8Q2J; Cell Signaling Technology), p-MAPK (9101; Cell signaling), MAPK (9102; Cell signaling), in Tween-PBS (TPBS), overnight at 4°C. Membranes were rinsed with TPBS and incubated with secondary antibodies (1:15,000); IRDye 800CW goat anti-mouse antibody or IRDye 680RD goat anti-rabbit antibody (Li-COR Biosciences), for 1 h while shaking at RT. Membranes were rinsed with TPBS and imaged using Odyssey Infrared Imaging System (Li-COR Biosciences, Nebraska United States), measuring 700 nm and 800 nm wavelengths. Protein-based densitometry to quantify p-MAPK/MAPK relative expression was performed using ImageJ (v1.53c).

Expression levels of Katnal2 proteins were determined by immunoblot analyses. Because Katnal2 protein levels in the brain were below the detection limit of immunoblotting using the currently available homemade Katnal2 antibodies (#2167; guinea pig polyclonal), which targeted the last 30 aa (aa 517–539) of the mouse Katnal2 protein, we used testis samples (8 weeks) prepared by tissue homogenization (150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl [pH = 7.4], 1% Triton-X100) and centrifugation followed by the use of the supernatant (not pellet).
For immunoblot analyses of choroid plexus lysates, 4 choroid plexus samples from 2 mice were pooled to make n of 1 and homogenized in RIPA buffer (10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.1 mM MgCl2, 1% NP-40 with protease inhibitor). The following antibodies were utilized for immunoblotting: aquaporin-1 (Sigma-Aldrich, AB2219; 1:1,000), Na⁺/K⁺-ATPase α1 (Sigma-Aldrich, 05–369; 1:1,000), AE2 (Santa Cruz; sc-376632; 1:1,000), and GAPDH (Cell Signaling, D4C6R; 1:1,000). Full-length immunoblot images can be found in S1 Raw Images.