Gapdh

N43/5 cells (3 × 10⁵) were cultured in 6-well plates and the day after, the cells were subjected to serum deprivation for 16 or 20 h. The cells were collected, lysed in RIPA buffer, and the GAPDH protein levels were evaluated by Western blot using GAPDH (Santa Cruz #sc-365062, CA, USA) and β-ACTIN antibodies.
N43/5 (2.5 × 10⁵) cells were plated in 6-well plates. The day after, the culture medium was changed to DMEM 2% FBS for 16 h and 100 μM PA or SA (or the same volume of BSA) was added for 6 h. Next, the cells were subjected to serum deprivation for 16 h. Finally, the cells were collected, lysed in RIPA buffer, and the levels of GAPDH evaluated by Western blot as described before.

Luteolin (purity > 98%, batch number: XC071225) was purchased from Xi'an Xiaocao Botanical Development Co., Ltd. (Xi'an, China). Sodium carboxymethyl cellulose (CMC-Na), prednisone, prednisolone, and dexamethasone (internal standard for HPLC analysis) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Methanol of HPLC grade was purchased from TEDIA Company Inc. (Beijing, China). The following items were purchased from the cited commercial sources: anti-11β-HSDI (Abcam, Cambridge, UK, dilution 1 : 800), anti-11β-HSD II (Abcam, Cambridge, UK, dilution 1 : 1000), GAPDH (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, dilution 1 : 500) antibodies, goat anti-rabbit IgG (HþL) HRP (BS13278), GAPDH (AP0063), and goat anti-mouse IgG (HþL) HRP (BS12478). All other reagents were of analytical grade.

Cell lysates were separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE). Primary antibodies used were: PRLR (D01PID5618, Abnova, Taiwan); Moesin (SC6410, Santa Cruz, Texas, USA); p-Moesin (SC12895, Santa Cruz, Texas, USA); FAK (SC271195, Santa Cruz, Texas, USA); p-FAK (SC11765-R, Santa Cruz, Texas, USA); c-Src (SC5266, Santa Cruz, Texas, USA); p-c-Src (SC166860, Santa Cruz, Texas, USA); GAPDH (SC59540, Santa Cruz, Texas, USA). The secondary antibodies Anti-rabbit (SC2357, Santa Cruz, Texas, USA) (1:3000) for PRLR; Anti-goat (SC2768, Santa Cruz, Texas, USA) (1:3000) for Moesin, p-Moesin and Actin, and Anti-Mouse (SC2005, Santa Cruz, Texas, USA (1:3500) for FAK, p-FAK, c-Src, p-c-Src, and GAPDH were incubated for 2 h in room temperature. Primary and secondary antibodies were incubated with the membranes, followed by three 5-min washings with TRIS-buffered saline-Tween 20. Immunodetection was carried out using enhanced chemiluminescence and was recorded with a quantitative digital imaging system (Quantity One, BioRad, Hercules, CA, USA), enabling us to assess saturation. Band densitometric analysis was quantified, as previously described (17 (link)), with the ImageJ image program (National Institute of Health – NIH, USA) using conditions ensuring analysis in the linear range of detection.

The distal end of nerve and middle part of gastrocnemius muscle was harvested two weeks after various treatment and proteins were extracted. Proteins (50 μg) were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto a blotting membrane. Antibodies against Bcl-2 (1:1000, BD), Bad (1:1000, Santa Cruz), Bax (1:1000, Santa Cruz), 8-oxo-dG (1:500, R&D), desmin (1:200, Proteintech), acetylcholine receptor (1:200, Millipore), GAPDH (1: 1000, Santa Cruz) were incubated overnight at 4°C. AFS and fibroblast cell lysated protein (50 μg) was separated by SDS-PAGE electrophoretically transferred to nitrocellulose membranes. After blocking, the blots were incubated with antibodies for NT-3 (1:1000, Millipore), BDNF (1:1000, Abcam), CNTF (1:1000, Abbiotec), GDNF (1:1000, Abbiotec), and GAPDH (1:1000, Santa Cruz). Horseradish peroxidase-conjugated secondary antibodies were used. Protein bands were analyzed by the ISI1000 image system (Alpha Innotech Corporation, CA, USA).