Anti gapdh

Cell lysis was performed with RIPA buffer (Beyotime, China). Protein was quantitated with BCA analysis (Beyotime, China). The protein extractions were separated with SDS/PAGE (10% gel) and transferred on to PVDF membranes (Sigma–Aldrich, U.S.A.). Subsequently, the membranes were incubated with primary antibodies (anti-E-cadherin, anti-N-cadherin, anti-ZEB2, anti-GAPDH) and with a secondary antibody (Cell Signaling Technology, U.S.A.). Subsequently, the membranes were incubated with primary antibodies: anti-E-cadherin (ab40772), anti-N-cadherin (ab76057), anti-ZEB2 (ab138222), anti-GAPDH (ab181602) and with a secondary antibody (#93702, 1: 2000 dilution, Cell Signaling Technology, CST, MA, USA). All primary antibodies were diluted at 1: 1000 and purchased from Abcam (Cambridge, MA, USA).The signals were measured by using a chemiluminescence system (Bio-Rad, U.S.A.) and analyzed with Image Lab Software.

Cellular or tissue proteins were extracted using a RIPA buffer containing 10% protease inhibitor (Roche) and quantified using a BCA kit. The proteins were then separated using a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were then probed with the primary antibodies, anti-RBPMS2 (1:1000, Abcam) and anti-GAPDH (1:2000, Cell Signaling Technology) at 4°C overnight. Finally, the membrane was incubated with secondary antibodies conjugated to horseradish peroxidase at 37°C for 60 min. Bands from immunoreactive protein bands were detected using electrochemiluminescence reagents and quantified using the ImageJ software. GAPDH acted as the internal reference.

Western blot was performed as previously described [29 (link)]. Briefly, the cells were lysed in radioimmunoprecipitation assay (RIPA) buffer consisting 2% protease inhibitor cocktail (Roche) and 1% phosphatase inhibitor (Roche) to obtain the protein. After that, BCA protein assay kit was used to test the protein concentrations. Thirty-five micrograms of total protein of each sample was subjected to SDS-PAGE. After electrophoresis, proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% nonfat milk and incubated with anti-AKR1C1 (Abcam, Cambridge, UK), anti-PPARγ, anti-RUNX2, anti-PGR, anti-AR, or anti-GAPDH (Cell Signaling Technology, Beverly, MA, USA) in Tris-buffered saline-Tween 20 (TBST) at 4 °C overnight. After that, the membrane was washed with TBST buffer, incubated with goat anti-rabbit IgG or goat anti-rat IgG (Abcam), and then washed with TBST again. At last, an ECL Western blot kit (CWBIO) was used to visualize the bands.

The EV pellets obtained from ultracentrifugation and cell pellets were lysed with 30 µL of boiling buffer with protease inhibitor. The protein concentrations were measured using a BCA protein assay kit. A total of 100 µg of protein lysates were mixed with 6× sample buffer and boiled at 95 °C for 10 min and then loaded into a gel (Novex WedgeWell 8 to 16%, Tris-Glycine, 1.0 mm, Mini Protein Gel, 12-well, Thermo Fisher Scientific, Waltham, MA, USA). The protein was transferred into a Pierce PVDF Transfer Membrane, 0.45 µm (Thermo Fisher Scientific, Waltham, MA, USA), blocked with 5% milk (Blotting-Grade Blocker, Bio-Rad, Berkeley, CA, USA) in TBST, and then incubated in the following primary antibodies overnight at 4 °C: anti-flotillin, anti-TSG101, anti-CD9, and anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA). After being washed with TBST for 10 min three times, the membranes were incubated in the AP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) for 3 h at 4 °C, washed three times, and exposed to Cytiva Amersham™ ECF™ Substrate (Thermo Fisher Scientific, Waltham, MA, USA) before visualizing the membranes using a Typhoon FLA 7000 biomolecular imager (GE Healthcare, Chicago, IL, USA).

Fermented Perilla frutescens (FPF), uracil, adenine, PCA, L7dGn, A7dGn and L7Gn were provided by Huons Co Ltd. (Seoul, Republic of Korea). Corticosterone, FXT and N-[2-[(hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl-benzo[b]thiophene-2-carboxamide (ANA-12) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Corticosterone enzyme-linked immunosorbent assay (ELISA) kit and dopamine ELISA kit were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Serotonin ELISA kit and ACTH ELISA kit were purchased from Abcam (Cambridge, UK). Anti-BDNF, anti-CREB, anti-pCREB, anti-ERK, anti-pERK, anti-TrkB, and anti-GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-pTrkB antibody was purchased from Abcam (Cambridge, MA, USA). All other chemical reagents were purchased from Sigma-Aldrich and were of analytical or HPLC grade.