P2714

The prepared brain slices were divided into the ACSF group and ACSF containing LPS 0.2 μg/mL group, both bathed for 3 hours or 6 hours. After an incubation that displayed significant β-APP accumulation, pre-treatment with RS-LPS (2.0 μg/mL) followed by incubation with LPS (0.2 μg/mL) or co-incubation with LPS+RS-LPS was performed for the same duration. Then, CC tissues were disseced and transferred to Tissue Extraction Reagent 1 (FNN0071, Invitrogen) with 1:1,000 protease inhibitor (P-2714, Sigma-Aldrich, St. Louis, MO, USA), and homogenized. Protein concentration was measured by bicinchoninic acid assay (BCA assay). Routine electrophoresis was carried out using 10% sodium dodecyl sulfate-polyamide gel. Polyclonal rabbit anti-β-APP antibody (1:600; #AB5302, Millipore, CA, USA) was used to detect β-APP. Mouse anti-β-actin (1:10,000, #A2228, Sigma-Aldrich) was applied as a gel loading control. Immunoreactivity bands were detected using enhanced chemiluminescence and developed with autoradiography film. Protein density of β-APP was measured using Image J and normalized to the corresponding β-actin in each sample.

C. reinhardtii was grown mixotrophically in Tris-acetate-phosphate (TAP) medium at 25 °C with vigorous shaking 90 rpm under 50 µmol photon m⁻² s⁻¹ photosynthetically active radiation^{75 (link)}. Cultures (50 mL, 5 replicates) from the exponential phase were centrifuged at 4,000 g for 15 min at 4 °C (Beckman Coulter Allegra® X-15R Centrifuge (Pasadena, CA, USA); rotor: 4750 A), then stored at −80 °C. Cells were broken by sonication in lysis buffer (15 mM Tris, 0.1 mM EDTA at pH 7.5) supplemented by protease inhibitors 40 µg mL⁻¹ (Sigma-Aldrich, P2714); the homogenate was centrifuged at 11,000 g, 4 °C for 30 min (2–16KC centrifuge using a 12132-H rotor, Sigma-Aldrich, Saint-Louis, MO, USA) to isolate the supernatant that mainly contained non-membrane proteins.

After screening, the cells were harvested and lysed in RIPA lysis buffer (Beyotime, China) which contained 1% proteasome inhibitor (P2714, Sigma) and 1 mM phenyl methane sulfonyl fluoride (PMSF) to detect the expression of Pgp. Equal amounts (25 μg) of protein were loaded onto SDS-PAGE for separation and then transferred onto a PVDF membrane. The membrane was blocked with 5% w/v nonfat dry milk (in TBS with 0.05% Tween-20). After incubation with Mdr-1 Antibody (mouse monoclonal IgG2b, Santa Cruze, USA) (1:1000) at 4°C overnight, the membrane was washed three times with TBST (TBS with 0.05% Tween-20) solution. The membrane was then incubated with secondary antibody (1:5000) at room temperature for 1 h followed by three washes with TBST. The protein bands were developed by incubation with a luminescent reagent. ImageJ was used to quantify the band intensity.

The rats were euthanized with isoflurane and brain slices were prepared as the same way as described in the Electrophysiology section. The CC was dissected from coronal brain slices in cold ACSF under a dissecting microscope and quickly transferred into Tissue Extraction Reagent 1 (FNN0071, Invitrogen, Camarillo, CA) with 1:1000 protease inhibitor (P-2714, Sigma). Protein homogenate concentrations were determined by bicinchoninic acid assay (BCA assay). After centrifugation, the supernatant was subjected to sodium dodecyl sulfate-polyamide gel electrophoresis and then the protein was transferred to a PVDF membrane. The membranes were then blocked with 5% non-fat milk and incubated with rabbit anti-β-APP antibody (1:500; Abcam USA), followed by HRP-conjugated secondary antibody (1:5000; Jackson Immuno Inc., West Grove, PA). Mouse anti-β-actin (1:10,000; Sigma) was used for the gel loading control. Protein bands were detected using enhanced chemiluminescence and developed with autoradiography film. Image J (NIH, Bethesda, MD) software was used to quantify the band densities and protein densities of β-APP values were normalized by corresponding β-actin in each sample.

We isolated cellular membranes from approximately 7 × 10⁷ idNs of WT and SCN1A^M145T. The procedure was similar to that already described in [18 (link)] for human tissues. The cells were scraped at day of differentiation 35, spun down and subsequently homogenized in membrane buffer (200 mM glycine, 150 mM NaCl, 50 mM EGTA, 50 mM EDTA, and 300 mM sucrose; plus 10 μL/mL of protease inhibitors, P2714 (Sigma)—pH 9 adjusted with NaOH). Then, the vials were centrifuged for 15 min at 9500× g. Afterwards, the supernatant was centrifuged for 2 h at 100,000× g with an ultracentrifuge. Finally, the pellet was resuspended in glycine 5 mM and used directly or aliquoted and kept at −80 °C for later usage.