Ha 51064 2 ap

Nuclear and cytoplasmic proteins were extracted with Nuclear and Cytoplasmic Protein Extraction Kit and quantified with a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturers’ instructions. PBMCs and total cell lysates were prepared in 1 × SDS buffer, directly analyzed by SDS-PAGE, and transferred onto nitrocellulose membranes. After blocking in 5% BSA in Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated with antibodies against Flag (F18804, Sigma, Inc.), HA (51064-2-AP, Proteintech), GLI3 (ab6050, Abcam), GLI1 (ab49314, Abcam), PTCH1 (ab53715, Abcam), SHH (ab32281, Abcam), GAPDH (ET1702-66, Huabio, Hangzhou, China), and β-actin (ET1701-80, Huabio). The antigen–antibody complexes were incubated with HRP-conjugated secondary antibodies (Bioworld, Nanjing, China) and visualized by an infrared imaging technique (Bio-Rad Laboratories, Inc.). The intensity of protein bands was quantified using ImageJ² to calculate the ratios of IntDen (GLI3)/IntDen (β-Actin) to ensure that the detection of protein bands was linearized. The Student’s t-test was used for the statistical comparison of GLI3 protein relative quantification between patients and normal controls.

The radio immunoprecipitation assay lysis buffer (RIPA) (P0013B, Beyotime, China) containing a protease-inhibitor cocktail (HY-K0010, MCE, USA) were used to extract proteins from cells. The proteins were lysed in SDS-loading buffer (FD006, Fdbio, China), then 20μg proteins were resolved on a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore, USA). The membrane was incubated with primary antibodies against PD-L1(13684, Cell Signaling Technology, USA), FLAG (80010-1-RR, Proteintech, USA), STT3A (12034-1-AP, Proteintech, USA), c-Jun (AF6089, Affinity, USA), HA (51064-2-AP, Proteintech, USA) and GAPDH (60004-1-Ig, Proteintech, USA) at a dilution of 1:1000, followed by incubation with species-specific (rabbit or mouse) HRP-conjugated secondary antibodies at a dilution of 1:5000. The immunoreactive bands were visualized by Omni ECL reagent (SQ101, EpiZyme, China).

Antibodies were as follows: AHSA1 (83036, Abcam, UK); HSP90 (13171-1-AP, ProteinTech Group, China); PSMD2 (14748-1-AP, ProteinTech Group, China); CDK6 (14052-1-AP, ProteinTech Group, China); HA (51064-2-AP, ProteinTech Group, China); MYC (16286-1-AP, ProteinTech Group, China); FLAG (F-4020, Merck KGaA, Germany); GAPDH (60004-1-Ig, ProteinTech Group, China); PARP (9542S, Cell Signaling Technology, USA); Caspase-3 (9662S, Cell Signaling Technology, USA); β-actin (4970S, Cell Signaling Technology, USA); Rabbit IgG (a7016, Beyotime Institute of Biotechnology, China) and mouse IgG (a7028, Beyotime Institute of Biotechnology, China).
Doxycycline (DOX) was purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Puromycin was purchased from Merck KGaA (Darmstadt, Germany). Bortezomib (BTZ) and Adriamycin (ADR) were purchased from Selleck Chemicals (Houston, TX). KU-177 was synthesized and characterized in Dr. Nianguang Li’s lab. Carfilzomib (CZ) was obtained from APExBIO Technology LLC (Houston, TX, US).

Human glioblatoma cell lines U251 and Human Embryonic Kidney (HEK) 293 cells were maintained in DMEM medium with high glucose and sodium pyruvate, supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). Human glioblatoma cell lines U87 were maintained in MEM medium supplemented with 10% fetal bovine serum. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air. Cells were authenticated that it origins from ATCC by short-tandem repeat profiling. Antibodies against HIF1α(20960-1-AP), GST(66001-1-Ig), and HA(51064-2-AP) were purchased from ProteinTech. Antibodies against FIH1(sc-26219) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). FLAG (F1804) was purchased from Sigma-Aldrich. Antibodies against CA9 (No. D120346), GLUT1 (No. D160433), LDH-A (No. D199841), and PHD2 (No. D122872) were purchased from Sangon Biotech. Antibodies against ATG5 (D5F5U), ATG7 (D12B11), ATG12 (D88H11), and LC3A/B (D3U4C) were purchased from Cell Signaling Technology.