Odyssey dual color infrared fluorescence imaging system

Co-IP and immunoblot analysis were performed, as mentioned previously [50 (link)]. In brief, for Co-IP, the cells were collected and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 1% Tris-HCl, and 10% glycerol) containing 1 mM PMSF and 1 × protease inhibitor cocktail (Basel, Switzerland, Roche). Then, cell lysates were incubated with anti-flag (M2) beads (Sigma-Aldrich, St. Louis, MO, USA) or added indicated antibodies and protein A+G Plus-Agarose (Santa Cruz Biotechnology, Dallas, TX, USA). After incubation 4–8 h at 4°C, the beads were washed three times with lysis buffer. For immunoblot analysis, the samples were separated by 10–12% sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinyl difluoride (PVDF) membrane (Sigma-Aldrich, St. Louis, MO, USA). After incubation with primary and secondary antibodies, the membrane is visualized by the Odyssey Dual Color Infrared Fluorescence Imaging System (LI-COR, Lincoln, NE, USA).

The DMH tissues of rats were homogenized mechanically in RIPA buffer mixed with a protease inhibitor cocktail (RW-0001) and phosphatase inhibitor (RP-WA0130). After centrifugation at 12,000 g at 4 °C for 5 min, the protein concentration was measured using a BCA quantification kit (RP-WA0201). Additionally, proteins were run on 8–10% polyacrylamide SDS-PAGE gels for electrophoresis (ZomanBio, ZD304C, Beijing, China), and subsequently transferred to polyvinylidene fluoride (PVDF) membranes using a Bio-Rad Transblot (Sigma-Aldrich, REF03010040001, Saint Louis, Missouri, USA). These membranes were blocked for 2 h in 5% skim milk/TBST or 5% bovine serum albumin/TBST at 37 °C and then incubated with antibodies against GAPDH (1:1000, Beyotime, China), β-tubulin (1:1000, Abcam, Cambridge, UK), GABAAR α1 (1:500, Sigma-Aldrich, Saint Louis, Missouri, USA), pSTAT3 (1:1000, HUABIO, Hangzhou, China), STAT3 (1:1000, HUABIO, Hangzhou, China), and CREM(C-2)/ICER (1:200, Santa Cruz) overnight at 4 °C. Then, the PVDF membranes were incubated with the corresponding fluorescent secondary antibody (LI-COR, 926-68071; Rockland, 610-145-002) at 37 °C, away from light, for 1 h. The signal was captured on an Odyssey dual-color infrared fluorescence imaging system (LI-COR, Lincoln, Nebraska, USA), and the protein bands were quantified and analyzed using ImageJ 1.52a software (NIH, Bethesda, MD, USA).