Immobilon western chemiluminescent hrp substrate system

Protein samples (5–10 µg) were separated on 8–10% gels by SDS-PAGE and transferred onto a nitrocellulose membrane (Merck, Millipore). The membrane was then blocked and incubated with the desired primary antibodies diluted 1:1000: anti OPCML (goat pAb, #AF2777, R&D Systems), phospho-AKT (Thr308 D25E6 XP® rabbit mAb, #13038, Cell Signaling), AKT (pan 11E7 rabbit mAb, #4685 Cell Signaling), phospho-ERK1 (pT202/pY204) + phospho-ERK2 (pT185/pY187) (mouse mAb, ab50011, Abcam), ERK1/2 (rabbit pAb, ab17942, Abcam) and anti-GAPDH (Cell Signalling), over-night at 4 °C. The membrane was washed, then incubated with the appropriate secondary antibodies (DAKO) diluted 1:5000. After final washings, proteins were detected using Immobilon Western Chemiluminescent HRP Substrate system (Millipore) and GE Healthcare Amersham™ Hyperfilm™ ECL film with a Kodak SRX2000 (Rochester, NY, USA) developer machine.

The total protein was extracted by using protein lysis buffer from the spinal cord tissues. The protein concentration was measured using BCA Protein Kit (Thermo Fisher). The protein sample (20 µg) was separated on 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The blots were probed with rabbit anti-SOCS3 (Abcam, Cambridge, U.K.; 1:200 dilution), rabbit anti-pSTAT3 (Abcam, 1:1000 dilution), rabbit anti-STAT3 (Abcam, 1:1000 dilution), rabbit anti-pJAK2 (Abcam, 1: 1000 dilution), rabbit anti-JAK2 (Abcam, 1:5000 dilution) for overnight at 4°C. The blots were incubated with horse radish peroxide-conjugated secondary antibody. The imaging was performed with electron chemiluminescence (ECL) emitting solution. Finally, the blots were visualized with an Immobilon Western Chemiluminescent HRP Substrate system (Millipore Corp., Billerica, MA, U.S.A.).

20 µg of cytoplasmic or nuclear fractions were obtained, and Western blots were performed by standard methods. Briefly, extracts were separated using 10% Bis-Tris NuPAGE gels (Thermo Fisher Scientific) and were electrotransferred to nitrocellulose membrane, which was blocked with 4% skim milk in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween-20 (TBST). GST and KLF3 proteins were probed for 1 hour in TBST using 0.2 µg/mL anti-GST antibody (1:5,000 dilution - ab19256, Abcam) or 0.2 µg/mL anti-KLF3 (1:5,000 dilution - PA5–18030, Thermo Fisher Scientific), respectively. KLF1 proteins were probed overnight in 4% skim milk in TBST using a KLF1 antibody that recognises both endogenous mouse KLF1 and GST-KLF1 DBD (1:1,000 dilution, antibody generation reported previously)^{20 (link)}. The Immobilon Western Chemiluminescent HRP Substrate System (Millipore Corporation) was used for detection. Membranes were subsequently stripped using 0.2 M NaOH for 10 minutes and were probed with anti-β-actin (1:30,000 dilution - A1978, Sigma) as a loading control.

The cells, which were cultured using same methods described for TIRF observation, were harvested and resuspended in 1 ml of sonication buffer (50 mM Tris-HCl, 200 mM NaCl, 10% glycerol). The precipitates were fractured via sonication (Bioruptor UCD-250; Cosmobio, Tokyo, Japan) at 250 W for 7 min. These fractions were used in an immunoblotting assay using a monoclonal anti-GFP antibody (Nacalai Tesque, Kyoto, Japan) following sodium dodecyl sulfate (SDS)-PAGE with a laboratory-produced 15% acrylamide gel. Immune complexes were detected using horseradish peroxidase (HRP)-labeled anti-mouse IgG (Cell Signaling Technology, MA., USA) and the Immobilon Western Chemiluminescent HRP Substrate system (Merck Millipore, MA., USA).

The overnight culture was diluted 100-fold into fresh TG medium and further cultured for 4 h. Immunoblotting using a monoclonal anti-GFP antibody (Nacalai Tesque). Immune complexes were detected using horseradish-peroxidase (HRP)-labelled anti-mouse IgG (KPL) and the Immobilon Western Chemiluminescent HRP Substrate system (Merck Millipore).

Protein was extracted from all cell lines using standard RIPA buffer methods. Protein concentrations were estimated with BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). SDS-Page was performed using the BioRad TGX system (Hercules, CA). Blots were blocked and incubated in primary antibody to LPA1 (Abcam) overnight, before incubation in a secondary HRP (Santa Cruz) followed by development with the Immobilon Western Chemiluminescent HRP Substrate system (EMD Millipore, Billerica, MA).