Bca protein assay reagent kit

Total cellular proteins were extracted using RIPA lysis reagent (Aspen) containing a protease inhibitor cocktail (Aspen) with or without a phosphatase inhibitor (Bio-swamp). The protein concentration was measured by BCA protein assay reagent kit (Aspen) and mixed with 4 × SDS loading buffer (Bio-swamp) for denaturation in a 100 °C boiling water bath for 8 min. The proteins were then separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Merck). The PVDF membranes were blocked with 5% skim milk for 90 min, washed three times, and then incubated with primary antibody at 4 °C overnight. Thereafter, the PVDF membranes were washed three times for 30 min and incubated with HRP-conjugated secondary antibodies for 90 min at room temperature. Finally, the membranes were again washed five times for 30 min, exposed to ECL developer (Aspen) and analyzed by Bio-Rad Image Lab software. The following primary antibodies used in this study were listed: Wnt5a (1:1000, Abcam), ERK1/2 (1:1000, Protein Tech), p-ERK1/2 (1:1000, CST), STAT3 (1:1000, Protein Tech), p-STAT3 (1:1000, CST), NF-κB p65 (1:1000, Protein Tech), p-NF-κB p65 (1:1000, CST), CaMKII (1:1000, Protein Tech), p-CaMKII (1:1000, CST), GAPDH (1:5000, Protein Tech), α-Tubulin (1:5000, Protein Tech).

Proteins were extracted from brain tissues and culture OPCs. The protein concentration was measured using a BCA protein assay reagent kit (Aspen, AS1086). As previously described,¹⁷ the protein lysates were separated by 4–12% Bis‐Tris sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE; Aspen, AS1012) and transferred to polyvinylidene fluoride (PVDF; Millipore, IPVH00010) membranes. Nonspecific binding sites were blocked by incubation in 5% nonfat dry milk in TBST buffer (20 mmoL/L Tris–HCl, 150 mmoL/L NaCl, and 0.05% Tween 20 (pH 7.5)) for 1 h. The membranes were then incubated at 4°C for 12 h with the following primary antibodies: rabbit anti‐Lef1 (1:1000; Proteintech, 14972‐1‐AP), rabbit anti‐P‐Smad1/5 (1:1000; Cell Signaling Technology, #9516), rabbit anti‐ACVR1 (1:2000; Abcam, ab155981), mouse anti‐MBP (1:1000; Santa Cruz, sc‐271524), and anti‐GAPDH (1:10000; Abcam, ab181602; as a control). After washing in TBST three times, the bound antibodies were detected by incubation with the corresponding secondary antibodies (1:10000; Aspen, AS1107) for 1 h at room temperature. The data were analyzed using an Odyssey Infrared Imaging System (LI‐COR Bioscience, Lincoln, NE, USA), and quantitative analysis was carried out using ImageJ software.

Sciatic nerves were dissected (n = 5) and frozen immediately in liquid nitrogen. Total protein was extracted, quantified using a bicinchoninic acid (BCA) protein assay reagent kit (ASPEN), and heated for 10 min at 95°C. Equal amounts of proteins were separated by 4%–12% Bis–Tris sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (Baiqiandu) and transferred to polyvinylidene fluoride membranes (Millipore). Nonspecific binding sites were blocked by incubation in 5% nonfat dry milk in TBST (Tris‐buffered saline, 0.1% Tween 20) for 1 h. Membranes were then incubated at 4°C for 12 h with primary anti‐Akt antibodies (1:2000; Cell Signaling Technology), anti‐phospho‐Akt (pAkt; 1:2000; Cell Signaling Technology), anti‐s6k (1:1000; Cell Signaling Technology), anti‐phospho‐s6k (ps6k; 1:1000; Cell Signaling Technology), anti‐p65 (1:1000; Abcam), anti‐phospho‐p65 (1:1000; Abcam), or control GAPDH (1:6000, Abcam). After three washes in TBST, bound antibodies were detected by incubation with the corresponding secondary antibodies (1:50,000) for 1 h. Data were analyzed using Image‐Pro Plus software, version 6.3 (Media Cybernetics, Inc., Rockville, MD, USA).