Ripa protein lysis buffer

The protein was extracted GBM cells by RIPA protein lysis buffer (Solarbio, R0010), with a freshly added mixture of protease inhibitor cocktail and PMSF. Then, 20 µg of protein was run on an SDS-PAGE gel, separated by electrophoresis and transferred to PVDF membranes. The membrane was first blocked by 5% BSA for 1-2 hours at room temperature, followed by incubation with primary antibodies at 4°C overnight. After washing three times with PBST, the membrane was blocked with secondary antibodies (Promega, 1:10000). Protein bands were detected was performed using G:BOXF3 (Syngene, UK) with Western HRP Substrate (Millipore, WBLUF0500). The antibodies used for western blots were: PD-L1 (Abcam, ab58810, 1:500), NF-κB (Cell Signaling Technology, 8242, 1:1000), Phospho-NF-κB (Cell Signaling Technology, 3033, 1:1000), UBXN1 (Proteintech, 16135-1-AP, 1:1000), GAPDH (Proteintech, 60004-1-Ig, 1:1000).

RIPA protein lysis buffer (Solarbio, Cat#R0020, Beijing, China) and protease inhibitor PMSF were used to extract total cell protein. The volume ratio of RIPA and PMSF is 100 μl:1 μl. We used the Bradford to measure the protein concentration at 595 nm. As for gel prepare, we used the SDS-PAGE Gel Kit (Solarbio, P1200-50T, China) to prepare 10% gel according to its manuscription. The prepared protein sample is added to the gel channel for constant voltage electrophoresis. After the electrophoresis, the protein in the SDS-PAGE gel is transferred to the PVDF membrane. Subsequently, the PVDF membrane was placed in 5% fat-free milk and blocked for 1 h at room temperature. Then the appropriate diluted primary antibody was used to incubate the PVDF membrane at 4 °C overnight. On the second day, wash the PVDF membrane three times with TBST for 10 min each time. After washing, the blots were incubated with HRP conjugated anti rabbit or antimice IgG for 1 h. The blots were developed in ECL mixture (Vector Laboratories, Burlingame, CA) and visualized by Imager. The corresponding antibody and dilution ratio are shown in the list A.

A RIPA protein lysis buffer (Solarbio) containing a protease inhibitor cocktail was used to isolate protein extracts from DPCs. Next, a BCA protein assay kit (ThermoFisher) was used to measure the absorbance at 562 nm to obtain protein concentrations. Total protein (20 µg) was subjected to SDS-PAGE, and the resolved proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Saint Louis, USA). The membrane was blocked in 5% bovine serum albumin (Solarbio) for 1 h and probed with primary antibodies overnight at 4 °C. The associated primary antibodies were those against alkaline phosphatase (ALP, ab224335, Abcam, Eugene, USA), versican (bs-2533R-A350, Bioss, Beijing, China), neural cell adhesion molecule (NCAM, bsm-52824R, Bioss), and β-catenin (ab32572, Abcam) and β-actin (ab8226, Abcam). After triple washing with Tris-buffered saline Tween (TBST, Solarbio), the membrane was incubated with the corresponding secondary antibody (ab150077, Abcam) for 1 h at 20–25 °C. An Odyssey infrared fluorescence scanning imager (Bio-Rad, Hercules, USA) was used for photography.