0.45 m polyvinylidene difluoride membrane

The cells or brain tissues were washed twice with cold PBS, then homogenized in a protein extraction buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors and centrifuged at 14,000 g at 4°C for 15 min. The supernatant was collected, and the protein concentrations were measured using the Bradford Protein Assay kit (Beyotime, Shanghai, China). After the protein sample loading buffer was added, the samples were boiled at 99°C for 10 min. The samples were then separated by 8% SDS-PAGE and transferred to 0.45 µm polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States). The membranes were blocked in 5% non-fat dry milk (diluted in the tris-buffered saline with 0.1% Tween 20 (TBST)) at room temperature for 1 h, then incubated in appropriate primary antibodies (diluted with 5% defatted milk) overnight at 4°C. Next, the membranes were washed three times for 10 min each with TBST and incubated with the secondary antibody (diluted with 5% defatted milk) at room temperature for 1 h. Finally, the membranes were detected using an enhanced chemiluminescent Plus Detection kit (Millipore, United States) and visualized using a Bio-Rad Imaging system (Bio-Rad, United States). This experiment was repeated in triplicate, and representative Western blot images were presented.

IPs were performed using the Nuclear Complex Co-IP Kit (Active Motif) under “stringent” conditions following the manufacturer’s instructions. For each IP, 10 µl of anti-Piwi mouse monoclonal antibody (Santa Cruz), 2.5 µl of anti-MEP-1 rabbit polyclonal antibody, or 2.5 µg equivalent of control IgG were added to 500 µg proteins (adjusted to 500 µl in IP buffer) and incubated overnight at 4 °C with rotation. Fifty microliters of Dynabeads protein G (Invitrogen) were then added and incubated at 4 °C for 2 h with rotation. The beads were then washed 6 times 5 min with 500 µl IP buffer and bound proteins were eluted in 30 µl of 2× Laemmli buffer (BioRad) at 95 °C. Western blotting was done following standard protocols. Proteins were separated using 4–15% TGX stain free polyacrylamide gels (BioRad) and transferred to 0.45 µm polyvinylidene difluoride membranes (Millipore). The membranes were blocked in 5% skimmed milk in TBS supplemented with 0.1% Tween 20 (TBST) for 1 h and incubated with primary antibodies overnight at 4 °C. After 3 washes in TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature (RT), followed by three washes in TBST.

RIPA lysis buffer (MCE, HY-K1001) and 10% protease inhibitor cocktail (MCE, HY-K0010) were used for total protein extraction of the mPFC (n = 4 per group) and colon (n = 4 per group). Protein concentrations were detected using the BCA Protein Assay Kit (Promega Biotech, Beijing, China) and a microtiter plate reader (Thermo Scientific, Waltham, MA, USA). For Western blotting, the proteins were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.45 µm polyvinylidene difluoride membranes (Millipore, Boston, MA, USA). All membranes were incubated with primary antibodies against TPH1 (CY5574, Abways, Shanghai, China), TPH2 (AY0821, Abways, Shanghai, China), SERT (382321, Zenbio, Chengdu, China), 5-HT_1BR (DF3497, Affinity, Golden, CO, USA), and GAPDH (ab8245, Abcam, Cambridge, MA, UK) at 4 °C overnight, followed by 1 h incubation at room temperature with secondary antibodies. The image was photographed using a Syngene GBox Imaging System (Gene Company, Shanghai, China) and an enhanced chemiluminescence solution (Millipore, Boston, MA, USA). Lastly, the expression level of the target protein bands was measured using Image Lab (version 3.0) software.

Cells were washed twice with cold PBS. Total protein was extracted by RIPA lysis buffer accompanied with 1 mM phenylmethylsulfonyl fluoride along with a proteinase inhibitor cocktail (Beyotime) and phosphatase inhibitor cocktail (Yeasen) and then quantified using the BCA Protein Assay kit (Beyotime). Western blotting assay was performed as described.^{55 (link)} Extracts of soluble cellular protein (20 µg) were separated by 10% SDS-PAGE and then transferred to a 0.45 µm polyvinylidene difluoride membrane (Millipore). After blocking with 5% bovine serum albumin for 1 h at 25 °C, each membrane was incubated with specific primary antibodies (Proteintech or Cell Signaling Technology) at 4 °C overnight. On the next day, each membrane was incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (Neobioscience) for 1 h at room temperature. Immunopositive bands were visualized via chemiluminescence (ECL reagent, Millipore) using a Model 4600 Chemiluminescence Imaging System (Tanon).