Ecl substrate solution

Cells were harvested and pelleted prior to lysis with lysis buffer containing 50 mM Tris-Cl, 150 mM NaCl, 0.5 % SDS, 1 % Triton X-100 with protease inhibitor cocktail (Roche). 50 μg of total protein lysates were resolved on 12 % SDS polyacrylamide gel (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Darmstadt, Germany), and probed with the following primary antibodies: caspase 3, Bcl-2, Bax, Bid (1:1000; all from Cell Signaling Technology Inc., Danvers, MA) and pan-actin antibody (1:10,000; NeoMarkers, Fremont, CA). Goat anti-mouse HRP-conjugated secondary antibody (1:10,000, Dako) or goat anti-rabbit HRP-conjugated secondary antibody (1:10,000, Dako) were used. All antibodies were diluted in blocking buffer [5 % bovine serum albumin (Sigma-Aldrich), 10 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.1 % Tween-20 (Merck)]. Protein bands were visualized using chemiluminesence by adding ECL substrate solution containing luminol in peroxide buffer (Thermo Scientific) to the membrane for 30s. Protein expression was quantified using MetaVue software (Molecular Devices, Sunnyvale, CA), normalized against actin levels.

iMDP-3 cells induced for various times (0, 1, 3, 5, 7, 11, and 14 days) were lysed with RIPA buffer (Santa Cruz Biotechnology) at 4 °C by vigorous shaking for 15 min. After centrifugation at 12,000g for 15 min, the supernatant was separated and stored at −80 °C until use. The protein concentration was measured by using a Bio-Rad protein assay kit (Bio-Rad Laboratory). An equal amount of proteins was loaded onto a 10 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE) system and the bands were subsequently transferred to a membrane (Bio-Rad Laboratory). Rabbit anti-Klf10 (Sigma-Aldrich) and goat anti-actin (Santa Cruz Biotechnology) were used as primary antibodies. Transferred proteins were incubated with ECL substrate solution (Thermo Scientific) according to the manufacturer’s instructions and labeled bands were revealed on X-ray film.

Spinal cords were homogenized in RIPA lysis buffer supplemented with complete proteinase inhibitor cocktail and PhoSTOP phosphatase inhibitors. Primary cells were lysed in RIPA buffer. Protein lysates were cleared of insoluble material through centrifugation, and the resulting protein lysates were subjected to SDS-PAGE. Proteins were wet transferred to 0.2 mm nitrocellulose membranes (BioRad Laboratories), which were blocked using 5% non-fat milk in 1% TBS-T buffer for 1 hr at room temperature. The membranes were incubated overnight using the following primary antibodies: anti-actin HRP, anti-Histone H3, anti-p65, anti-pp65, anti-STAT1, anti-pSTAT1, anti-ZAP70, anti-pZAP70, anti-PLC-g1, anti-pPLC-g1, anti-NFAT1, anti-mTOR, anti-pmTOR, anti-AKT, anti-pAkt, anti-Akt, anti-4EBP1, anti-p4EBP1, anti-S6K, anti-pS6K, anti-c-Myc, anti-pc-Myc, anti-JNK, anti-pJNK, anti-ERK½ or anti-pERK½. All primary antibodies were used at a 1:1,000 dilution in 5% non-fat milk. Membranes were washed in TBS-T and incubated with the following appropriate secondary antibodies: goat anti-rabbit-HRP or goat anti-mouse HRP. All secondary antibodies were used at a 1:40,000 dilution in 5% non-fat milk. Protein bands were visualized following exposure of the membranes to ECL substrate solution (ThermoFisher) and quantified by densitometryc analysis using ImageJ software.