Horseradish peroxidase conjugated secondary antibodies

Spinal cord samples were lysed using ice-cold RIPA buffer with protease inhibitors (Beyotime, China), followed by centrifugation at 12,000 g/min for 30 min at 4 °C. Supernatants were collected, and protein concentrations were determined with the BCA protein quantitative analysis kit (Beyotime, Shanghai, China). After the addition of a loading buffer and thermal denaturation at 99 °C for 5 min, proteins were separated using 8% sodium dodecyl sulfate–polyacrylamide (SDS-PAGE) gels and transferred to PVDF membranes (Millipore, Bedford, MA, USA) at 120 mA for 60 min. Membranes were blocked with 5% nonfat milk for 2 h at room temperature and were incubated with a rabbit anti-SGK3 antibody (1:1000, Servicebio, Wuhan, China) and rabbit anti-GAPDH antibody (1:1000, Proteintech, Rosemont, IL, USA) at 4 °C overnight. Following three washes with TBST for 10 min, membranes were incubated with the horseradish-peroxidase-conjugated secondary antibodies (1:5000, Absin, Shanghai, China) for 1 h at room temperature. Immunoreactive proteins were visualized using super ECL Western blot detection reagents (Millipore, Burlington, MA, USA). The intensity of each band was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to that of GAPDH.

One hundred milligrams of tissue were homogenized for extracting proteins in 1 mL ice-cold RIPA buffer (50 mM Tris-HCL pH 7.4, 150 mM NaCl, 1% NP, 0.1% SDS, 1 × phosphatase/protease inhibitors). The protein concentration was determined using the BCA protein assay kit (Beyotime, Nantong, China). Western blotting was performed as described previously [20 (link)]. About 20 μg of protein from each sample was separated on 10% SDS-PAGE and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked in TBS buffer containing 5% bovine serum albumin (20 mM Tris–HCl (pH 7.6) and 150 mM NaCl) for 1.5 h at 37 °C. Next, these blots were incubated overnight at 4 °C with rabbit anti-GAPDH, rabbit anti-MEK1/2, rabbit anti-p-MEK1/2, rabbit anti-Bax, rabbit anti-Bcl-2, and rabbit anti-Rasd1 using the dilutions listed in Table 1. Blots incubated with rabbit IgG served as negative controls. The blots were then incubated with species-matched horseradish peroxidase-conjugated secondary antibodies (Absin, Shanghai, China). The chromogenic signal intensity was detected using an Odyssey Scanning System (LI-COR, Lincoln, NE, USA) and quantified using image J software (NIH, Bethesda, MD, USA).

The OB and SN were collected and lysed in were lysed with RIPA lysis buffer (Beyotime, China) and protease inhibitor (Roche, USA). The protein concentration was determined using the BCA protein assay kit (CWBIO, USA) and. The samples of 15 μg were electrophoresed and separated on 10% sodium dodecyl sulfate-polyacrylamide gel. The proteins were then transferred to polyvinylidene difluoride membrane (Millipore, USA) for 90 min. After blocking for 2 h with 5% non-fat milk in TBST at room temperature (RT), the membranes were incubated with primary antibodies against TH (1:3000, Millipore, USA), L-ferritin (1:1000, Abcam, UK), glutamic acid decarboxylase (GAD, 1:10000, Abcam, UK), GAPDH (1:20000, absin, China), β-actin (1:10000, Bioss, China) overnight at 4 °C. After washing in PBS, the membranes were incubated with horseradish peroxidase–conjugated secondary antibodies (1:10000, absin, China) were applied for 1 h at RT. Immunoreactive bands were detected and visualized with Fusion system (UVP, USA). The band intensities were normalized to those of β-actin or GAPDH.