A9169

Western blotting was conducted according to our previous descriptions (Li et al., 2018b (link)). Briefly, hippocampal tissues were lysed in high KCl lysis buffer containing 10 mM Tris–HCl, pH 8.0, 140 mM NaCl, 300 mM KCl, 1 mM EDTA, 0.5% Triton X-100, and 0.5% sodium deoxycholate with 1 mM phenylmethylsulfonyl fluoride (Roche, United Kingdom). The supernatant was quantified using a commercial BCA kit (Beyotime Biotechnology Institute, China). Protein samples were separated by SDS-polyacrylamide gels (SDS-PAGE) and transferred electrophoretically to polyvinylidene fluoride membranes. After blocking, the membranes were incubated with 4-hydroxynonenal (4-HNE) (Mouse, 39–122 KDa, MAB3249, 1 μg/ml, Novus, United Kingdom), GPX4 (Rabbit, 22 kDa, ab125066, 1:5000, Abcam, United Kingdom) and β-actin (Mouse, 43 kDa, A5441, 1:10000, Sigma-Aldrich, United States) overnight. The next day, after washing, the membranes were then incubated with secondary IgG goat anti-rabbit (A9169, 1:10000, Sigma-Aldrich, United States) or rabbit anti-mouse antibody (A9044, 1:10000, Sigma-Aldrich, United States). Immunodetection was performed using an enhanced chemiluminescence kit and the intensity of protein bands was analyzed by Quantity One software (BioRad, United States).

Protein extraction from the cells was achieved using Triton lysis buffer supplemented with protease and phosphatase inhibitor mixtures (Sigma). Cell lysates were loaded with Laemmli buffer, centrifuged for 10 min, and boiled for 5 min. Proteins were separated by SDS-PAGE using 10% SDS-polyacrylamide gel and transferred in a wet condition onto a nitrocellulose membrane (Amersham Biosciences). After blocking with 5% nonfat dry milk, proteins were labeled overnight with primary antibodies as follows: goat anti-NRX (R&D Systems, AF5719; dilution 1:2000); rabbit anti-DVL2 (Cell Signaling, 3216; dilution 1:500); and rabbit anti-GAPDH (Santa Cruz Biotechnology, sc-25778; dilution 1:800) used as stable housekeeping protein. Proteins were probed for 1 h with HRP-conjugated secondary antibodies as follows: anti-goat (Santa Cruz Biotechnology, sc-2020; dilution 1:50,000) and anti-rabbit (Sigma, A9169; dilution 1:80,000). Signals were detected with ECL Western blot detection reagent (GE Healthcare) and quantified with Fiji/ImageJ. Relative signal intensities were normalized to GAPDH.

Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were incubated with anti-PER2 antibody (Beckton-Dickinson, GP81620-050) 1:1000, anti-p53 antibody (Oncogene, OP03) 1:1000, or anti-actin antibody (Sigma, A5060) 1:1000 (all diluted in blocking buffer) at 4°C over night. They were washed and subsequently incubated with appropriate HRP-conjugated secondary antibodies (anti-rabbit for actin, anti-mouse for PER2 and p53; both Sigma, A9169 and A9044) for 1 h at RT. Detection was performed using the Western blotting detection reagents kit (Amersham Biosciences, 1059243) according to manufacturer’s instructions. Membranes were exposed on Hyperfilm (Amersham Biosciences, RPN1678K). For quantitative analysis, bands were quantified (QuantityOne 3.0, Biorad), and band intensity was normalized to actin.

Samples destined for Native-PAGE analysis were combined with native sample buffer (50 mM Tris-HCl, pH 7.4, 10% glycerol, 0.0014% xylene cyanol at final concentration) and loaded onto polyacrylamide gels [90 mM Tris-borate, pH 8.35, 5 mM MgCl₂, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 2.5% sucrose] with 3% stacking and 4% resolving portions. Electrophoresis was performed with native running buffer (90 mM Tris-borate, pH 8.35, 5 mM MgCl₂, 0.5 mM EDTA) at 100 V for 3 h on ice. Samples prepared with 20 mM ATP were separated using gels and running buffer containing 1 mM ATP. Samples intended for SDS-PAGE analysis were combined with Laemmli sample buffer [39 (link)] and resolved on 12% gels. Where necessary, gels were stained with Coomassie Brilliant Blue R-250 or silver nitrate [13 (link)]. Alternatively, proteins resolved by Native-PAGE or SDS-PAGE were transferred onto nitrocellulose membrane (PALL BioTrace™; 66,485) and immunoblotted with specific antibodies. Antibodies used in this study include anti-FLAG (Sigma; F1804), anti-PBA1 (Abcam; ab98861), anti-RPN1 (Abcam; ab98865), anti-HSP70 (Agrisera; AS08 371), anti-PA200 (Agrisera; AS19 4269), goat anti-mouse conjugated to horseradish peroxidase (HRP) (Cell Signaling; 7076S), and goat anti-rabbit conjugated to HRP (Sigma; A9169). Chemiluminescent signals were quantified using ImageJ (https://imagej.nih.gov/ij).

Cells were pretreated with darifenacin (0.1, 1 and 10 µM) for 60 min, followed by addition of ACh (10 µM) for 5 min. Following treatment with ACh, darifenacin or both, cells were lysed using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific) containing phosphatase and protease inhibitors. Protein concentration was quantified by a Quantipro BCA protein assay kit (Sigma-Aldrich) according to the manufacturer’s protocol. Cell extracts were separated by polyacrylamide gel electrophoresis and blotted on polyvinylidene fluoride membranes (Perkin Elmer, Boston, MA, USA). For immune detection, the following primary antibodies were used: anti-p38 (8690), anti-phospho-p38 (4511), anti-ERK1/2 (4695), anti-phospho-ERK1/2 (4370), anti-Akt (pan, 4685), anti-phospho-Akt (pan, 9018), anti-Src (2109S), anti-phospho-Src (6943S; all antibodies 1:1000, Cell Signaling, Leiden, Netherlands) and anti-β-actin (A5441, Sigma-Aldrich). Bound antibodies were visualized by luminescence imaging (Fusion FX, Vilber, Marne-la-Vallée, France) using peroxidase-conjugated secondary antibodies (A9169 and A9044, Sigma-Aldrich) and a chemiluminescent substrate system (SuperSignal West Pico PLUS, Thermo Fisher Scientific). Densitometric quantification was carried out with Image J. β-actin served as internal loading control.