Gapdh d4c6r

Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors for 20 min. Proteins (20 μg) extracted from cells were separated on SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked for 1 h with blocking buffer (5% non-fat milk in PBS containing 0.1% Tween 20) and then incubated with primary antibodies dissolved in blocking buffer at 4 ℃ overnight. Membranes were washed with TBS-T buffer (PBS with 0.1% Tween 20) for 5 min three times and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h. The membrane was developed using an ECL Kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) with X-ray film.
The primary antibodies used in this western blotting were as follows: E-cadherin (H-108, 1:1000, sc-7870; Santa Cruz Biotechnology, Inc.), N-cadherin (H-63, 1:1000, sc-7939; Santa Cruz Biotechnology, Inc.), fibronectin (EP5, 1:1000, sc-8422; Santa Cruz Biotechnology, Inc.), vimentin (V9, 1:1000, sc-6260; Santa Cruz Biotechnology, Inc.), slug (1:1000, GTX31749; GeneTex), snail (1:10000, GTX125918; GeneTex), and GAPDH (D4C6R, 1:1000, 97,166; Cell Signaling Technology).

Whole protein was isolated by using whole-cell lysis buffer in the presence of a protein phosphatase inhibitor cocktail (Pierce, USA). For detecting the YAP nuclear translocation, Nuclear-cytoplasmic fractionation was conducted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol, which enable stepwise separation and preparation of cytoplasmic and nuclear extracts from Schwann cell lineage. After the samples were electrophoresed through 8-12% polyacrylamide gels, proteins were transferred onto a PVDF membranes. The membranes were incubated with primary antibodies. Antibodies include (Cell Signaling Technology, USA, unless otherwise indicated): NF1 (D7R7D) (1:1,000, #14623) YAP (D8H1X) (1:1,000, #14074), p-YAP (S127) (D9W2I) (1:500, #13008S), β-Actin (13E5) (1:1,000, #4970), Cleaved Caspase-3 (Asp175) (1:1,000, #9664), GAPDH (D4C6R) (1:1,000, #97166) and Histone H3 (1:1,000, affinity # BF9211). After incubated with peroxidase-linked secondary antibodies (GK500705, Gene Tech, China), the membranes were washed and visualized using Immobilon Western HRP Chemiluminescence Substrate (Millipore, USA).

We harvested and lysed cells with 5 × loading buffer (Beyotime, Beijing, China) after culture for 48 h. We fully lysed the cell lysates with a vortex mixer (Thermo Fisher, Waltham, MA, United States) at 3000 rpm. The protein samples were then incubated in a dry bath incubator (Thermo Fisher, Waltham, MA, United States) at 100°C for 10 min to denature the proteins. We subjected protein samples to SDS–PAGE for gel electrophoresis, transferred the gel to PVDF membranes (Millipore, Boston, MA, United States) for transfer electrophoresis, and then exposed them by ECL (Thermo Fisher, Waltham, MA, United States). We purchased primary antibodies against VDR (D2K6W) and GAPDH (D4C6R) from Cell Signaling Technology (CST, Boston, MA, United States). We obtained HRP-conjugated antibody (ZB-2301, ZB-2305) from ZSGB-Bio Company (Beijing, China). All experiments were repeated at least three times.