Wet transfer cell

A minimum of 500,000 cells were used per assay. Similar to previously published protocols [24 (link)], the cells from CYP1B1-edited microglia and astrocytes were lysed, and the proteins were quantified. Likewise, the mock-edited microglia and astrocytes were lysed, and the proteins were quantified using a Microplate BCA Protein Assay Kit. Immunoblot analysis was performed by loading 20μg of protein samples on an SDS page of 10–20% gels (Life Technologies). Transfers were performed on wet transfer cells (Bio-Rad) with PVDF membranes (EMD Millipore). The antibodies used were anti-β-actin, internal control (Catalogue #3700, Cell Signaling Technology, USA), RXRα (Catalogue #3085, Cell Signaling Technology, USA), RXRβ (Catalogue #8715, Cell Signaling Technology, USA), RXRƳ (Catalogue #5629, Cell Signaling Technology, USA) and melatonin (MTNR1B) (Catalogue #ab203346, Abcam, USA). The secondary antibodies were goat anti-mouse (Catalogue #926–32210, Li-Cor, USA) and goat anti-rabbit (Catalogue #926–68021, Li-Cor, USA).

Caco-2 cells were treated with 5 mM β-xyloside or 10 µM Stattic for 4 h at 37 ℃, and then were incubated with GST-rTsCTL (5 µg/mL) at 37 ℃ for 2 h [17 (link)]. The Caco-2 cells were lysed in RIPA buffer, ultrasonicated in an ice bath for 30 s and centrifuged at 12 000 × g for 15 min to remove any cell fragments. The cell soluble proteins were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore, USA) in the wet transfer cell (Bio-Rad, USA). The membrane was blocked with 5% skim milk in TBST at 37 °C for 2 h and incised into strips. The strips were probed with antibodies against syndecan-1 (1:1000), total STAT3 (t-STAT3; 1:1000), phosphorylated STAT3 (p-STAT3; 1:5000, Abmart, China), occludin (1:500), claudin-1 (1:200), claudin-2 (1:200) (ThermoFisher, USA), and anti-β-Actin antibody (1:1000) overnight at 4 °C [6 (link)]. After washes with TBST three times, the strips were incubated at 37 °C for 1 h with HRP-conjugated anti-rabbit IgG (1:10 000; Southern Biotech). And then, Omni-ECL reagents (Epizyme, Shanghai, China) were used to visualize the reactive bands, and the relative intensities of each band were analyzed using the Image J software (National Institutes of Health, USA) [36 (link)].

The purified rTsCTL were separated on 10% SDS-PAGE [35 (link)]. The proteins were transferred onto nitrocellulose (NC) membrane (Millipore, USA) in the wet transfer cell (Bio-Rad, USA) [36 (link)]. The membrane was blocked using 5% skimmed milk in Tris-buffered saline containing 0.05% Tween (TBST) at 37 °C for 2 h, and cut into strips. The strips were incubated with diverse serum (1:100; anti-rTsCTL serum, infection serum, anti-GST serum and normal serum) at 37 °C for 2 h. After being washed using TBST, the strips were incubated at 37 °C for 1 h with HRP conjugated-anti-mouse IgG (1:10 000; Southern Biotech, USA). After washes, the color was developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich) [37 (link), 38 (link)].

Accessory glands were dissected from 3–5 day old virgin males, placed into tubes with SDS-sample buffer, homogenized, and boiled for 5 min. Extract representing the equivalent of one male’s accessory glands was loaded onto 12% or 4–20% SDS-PAGE gels, purchased from (Invitrogen/Thermo Fisher Scientific). The proteins were run slowly (20–30 volts) through the stacking gel and first half of the separating gel. Later, when the proteins were in the separating gel, the voltage was turned up to at 100V. Transfer of the proteins onto PVDF membranes was performed using a Biorad wet transfer cell for 1 hour at 100V. Antibodies used for the western blots were affinity purified rabbit anti-CG1656 (1:500 dilution) [74 (link)], affinity purified rabbit anti-CG1652 (1:250) [74 (link)] and rabbit anti-Acp36DE (1:30000)[75 (link)]. Secondary antibodies were goat anti-rabbit antibodies conjugated to either alkaline phosphatase (Biorad) or horseradish peroxidase (Promega). Signals for the alkaline phosphatase conjugated antibodies were visualized according to the NBT/BCIP staining kit (Roche), while peroxidase conjugated antibodies were visualized according to the Supersignal West Pico PLUS Chemiluminescent Substrate kit (Invitrogen/Thermo Fisher Scientific). PGNase treatments of AG protein extracts were performed as in Gligorov et al. {Gligorov, 2013 #176}

Whole-cell lysates were prepared in 2× Laemmli sample buffer. Proteins were separated by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis, on 7% gels (26 ). The proteins were transferred to a PVDF membrane in a wet transfer cell (Bio-Rad) using a buffer containing 25 mM Trizma base and 192 mM Glycine. After transfer the proteins were fixed to the membrane in 100% methanol for 5 min. The membranes were blocked in 5% non-fat milk before probing with the relevant antibody: γ-tubulin, diluted 1:10 000 (Sigma-Aldrich T6557), RB1 1:500 (BD Biosciences 554136), RB1-phospho (ser795) 1:1000 (Abcam ab47474). Detection was performed using an enhanced chemiluminescence kit (Amersham Biosciences) and quantified in a Chemi Genius (Syngene) with the software GeneTools.