Westsave gold western blot detection kits

T-MSCs and T-MSC-SCs were washed with ice-cold PBS and lysed in PRO-PREP buffer containing a phosphatase inhibitor cocktail solution (iNtRON Biotechnology, Seongnam-si, Korea) for 30 min on ice. After centrifugation at 13,000× g for 20 min at 4 °C, equal quantities of protein from supernatants were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and were electrophoretically transferred onto polyvinylidene membranes (Millipore, Billerica, MA, USA). The blots were then probed overnight at 4 °C with antibody against the glial fibrillary acidic protein (GFAP) (1:400, monoclonal antibody, Sigma-Aldrich, cat. no. G3893) or the nerve growth factor receptor (NGFR/p75) (1:500, polyclonal antibody, Santa Cruz Biotechnology, cat. no. sc8317, Dallas, TX, USA), followed by the corresponding secondary antibody. The blots were washed and developed using enhanced chemiluminescence reagents (WestSave GOLD™ Western Blot Detection kits) (AbFrontier, Seoul, Korea), according to the manufacturer’s instructions. Band intensities were assessed by densitometric scanning (LAS-3000, Fujifilm, Tokyo, Japan).

Protein samples from total cell extracts were washed with ice-cold phosphate-buffered solution (PBS). Protein samples were lysed in Pro-Prep buffer containing a phosphatase inhibitor cocktail solution (iNtRON Biotechnology, Seongnam-si, Korea) for 30 min on ice followed by centrifugation at 13,000× g for 20 min at 4 °C. Equal amounts of protein from each supernatant were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred onto polyvinylidene membranes (Millipore, Billerica, MA, USA). Then the blots were probed overnight at 4 °C with monoclonal antibodies against Islet 1 (Cat. no. ab86472), HB9 (Cat. no. ab79541), or ChAT (Cat. no. ab178850) (1:500, all from Abcam, Cambridge, UK) followed by the corresponding secondary antibody. The blots were developed using enhanced chemiluminescence reagents (WestSave Gold Western Blot Detection kits; AbFrontier, Seoul, Korea). The intensity of each band was assessed by densitometric scanning (LAS-3000; Fujifilm, Tokyo, Japan). The expression level of each protein was normalized with the respect of GAPDH levels (No. LF-PA0018; 1:1000, pRb, AbFrontier). The protein levels were quantified using Multi Gauge software (version 3.0; Fuji Photo Film, Kanagawa, Japan).

Sciatic nerve tissues were washed with ice-cold PBS and lysed in Pro-Prep buffer containing a phosphatase inhibitor cocktail solution (iNtRON Biotechnology, Seongnam-si, Korea) for 30 min on ice. After centrifugation at 13,000× g for 20 min at 4 °C, equal quantities of protein from supernatants were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and were electrophoretically transferred onto polyvinylidene membranes (Millipore, Billerica, MA, USA). The blots were then probed overnight at 4 °C with antibody against v-Akt Murine Thymoma Viral Oncogene (Akt; No. 9272) or Phospho-Akt (p-Akt; Thr308; No. 9275) or extracellular-signal-regulated kinase (ERK; Phospho-p44/43 MAPK (Erk1/2) (Thr202/Tyr204); No. 9101) (1:500, all pRb, Cell signaling) followed by the corresponding secondary antibody. The blots were washed and developed using enhanced chemiluminescence reagents (WestSave Gold Western Blot Detection kits; AbFrontier, Seoul, Korea), according to the manufacturer’s instructions. Band intensities were assessed by densitometric scanning (LAS-3000, Fujifilm, Tokyo, Japan). The p-Erk protein expression level was normalized to the expression of GAPDH (No. LF-PA0018; 1:1000, pRb, Ab Frontier, Korea). The results were quantified using Multi Gauge software, version 3.0.