Gar007

Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Roche, Mannheim, Germany, 11873580001). Protein concentration was determined. Equal amounts (20 ug/lane) of proteins were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Roche, 03010040001). After blocking with 5% nonfat milk in Tris-buffered saline for 1 h, the membrane was incubated overnight at 4 °C with specific primary antibodies, followed by incubation with appropriate HRP conjugated secondary antibodies. Signals were detected using an enhanced chemiluminescence reagent (Millipore, Darmstadt, Germany, WBKLS0500) and subjected to Alpha Innotech Flour Chem-FC2 imaging system (Alpha Innotech, San Leanardo, CA). The antibodies used in WB were as follows: anti-RTN3 (Abcam, ab68328, 1:1000 dilution), anti-SOCS2 (Abcam, ab3692, 1:1000 dilution), anti-UPB1 (Abcam, ab157195, 1:1000 dilution), anti-β-Actin (Cell Signaling Technology, #4970, 1:1000 dilution), horseradish peroxidase (HRP)-conjugated secondary antibodies (Multisciences, GAR007 and GAM007, 1:2000 dilution). Quantitative densitometry was performed using Photoshop CC (Adobe, CA, USA).

Cells were blocked with Dako Protein Block (Dako, Mississauga, ON, Canada) as previously described^{18 (link)}. The AQP2 antibody (1:100 diluted in Dako Protein Block, DF7560, Affinity Co., Cincinnati, OH, USA), the corresponding secondary donkey anti-rabbit IgG H&L (1:100 diluted in Dako Protein Block, Alexa Fluor 647), another AQP2 antibody (1:500 diluted in Dako Protein Block, Ab62628, Abcam Co., USA), the corresponding secondary goat anti-rabbit IgG H&L (1:10,000 diluted in Dako Protein Block, BL003A, Biosharp), anti-ERα (1:100 diluted in Dako Protein Block, Proteintech, 21244.1.AP), rabbit anti-ERβ mouse (1:100 diluted in Dako Protein Block, Santa Cruz Sc-53494), and horseradish peroxidase-conjugated secondary antibodies (MultiSciences GAR007, GAM007) were used. Finally, the cells were counterstained with the chromosomal dye 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime Biotechnology, C1006).

Tissue lysates were prepared using testes from C57BL/6 mice at various ages. Briefly, testes were first extracted from mice and removed of tunica albuginea. They were then briefly homogenized on ice using a glass homogenizer in a lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.2) containing 1% Triton X-100, 1X Protease Inhibitor Cocktail (Roche, 04693132001, Schaffhausen, Switzerland). The tissue homogenates were further incubated by rotating slowly at 4 °C for 2 h. Undissolved materials were discarded after centrifugation at 12,000 rpm and 4 °C for 15 min using a refrigerated tabletop centrifuge (Eppendorf, 5427R, Enfield, CT, USA). The supernatants were transferred into fresh Eppendorf tubes, and protein concentrations were measured by spectrometry. Western blotting was done according to standard procedures after proteins were separated using SDS-PAGE. The primary antibodies used were: mouse monoclonal anti-MENA (Santa Cruz Tech., sc135988, Dallas, TX, USA, 1:2000), and rabbit polyclonal anti-α-TUBULIN (Proteintech, 11224-I-AP, Rosemont, IL, USA, 1:5000). The secondary antibodies used were: goat-anti-mouse-HRP (Multisciences, GAM007, Hangzhou, China) and goat-anti-rabbit-HRP conjugates (Multisciences, GAR007, Hangzhou, China).