Pierce ripa buffer

The cells were collected in Pierce^TM RIPA buffer (89901, Thermo Fisher Scientific) supplemented with 3% phosphatase and protease inhibitors (78442, Thermo Fisher Scientific) on ice and incubated for 30 min. Protein concentration was measured using the Pierce^TM BCA Protein Assay Kit (23225, Thermo Fisher Scientific) on a Nanodrop 2000 Spectrophotometer. Protein lysates (20 μg) were separated by electrophoresis on 4-20% Mini-PROTEAN^@ TGX^TM Precast Gel (#4561093, Bio-Rad) and transferred to a nitrocellulose membrane (1704271, Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad).
Membranes were blocked with 5% non-fat milk solution (T145.1, ROTH) and then incubated for 1 h at RT employing either PD-L1 polyclonal antibody (PA5-20343, Invitrogen) (dilution 1:1000) or HIF-1α polyclonal antibody (PA1-16601, Invitrogen) (dilution 1:1000). β-actin (ab8227, Abcam) (dilution 1:2000) was used to standardize the loading. Polyclonal Goat Anti-Rabbit (P0448, Dako) HRP at a dilution 1:2000 was used as secondary antibody. The signals were detected utilizing Amersham ECL Western Blotting Detection Reagents (9838243, GE Healthcare, Freiburg, Germany) on Amersham Hyperfilm (28906836, GE Healthcare) on OPTIMAX X-Ray Film Processor (11701-9806-3716, PROTEC GmbH, Oberstenfeld, Germany). The ImageJ program (version 2, NIH, Washington, DC, USA) was used for densitometric analysis.

To extract proteins, hNSCs were collected and lysed in PierceTM RIPA buffer (Thermo, 78510, Waltham, MA, USA) (100X Phosphatase Inhibitor Cocktail, GRF101.100X Protease Inhibitor Cocktail, GRF102). The Bradford method was used to quantify the lysates before equal amounts of proteins were added for the western blot assay. Antibodies used for the western blot were anti-SPT16 (Abcam, ab204343, 1:2000, Cambridge, UK), anti-PCNA (Abcam, ab92552, 1:2000), anti-phospho-mTOR (Ser2448) (CST, Cat#5536T, 1:2000), anti-phospho-AKT (S473) (CST, Cat#4060S, 1:1000), anti-AKT (CST, Cat#4691S, 1:2000), anti-PAX6 (Abcam, ab195045, 1:2000), anti-LC3B (Abcam, ab192890, 1:2000), and anti-SOX2 (Abcam, ab92494, 1:2000). Before being transferred to a PVDF membrane, the proteins were separated using 12% SDS-PAGE (BIO-RAD, TGX Stain-Free Fast Cast Acrylamide Kit 12% Cat#1610185) for 2 h. After incubation with QuickBlock^TM (Beyotime, P0252) for 30 min at room temperature. The primary antibodies were incubated with the membrane at 4 °C for the whole night. The membranes were then subjected for two hours at room temperature to HRP-conjugated goat anti-rabbit IgG (GtxRb-003-DHRPX, immunoReagents, 1:5000). At least three different western blot studies were conducted. Using ImageJ software (https://imagej.nih.gov/ij/docs/guide/146-30.html accessed on 1 March 2022), the western blot bands were quantified.

RAW 264.7 macrophages were treated as same as RT-PCR. The cells were gently washed twice with ice-cold PBS, and lysed in Pierce^TM RIPA Buffer (Thermo Fisher, San Diego, CA, USA). Proteins were collected by centrifugation at 4 °C, 12,000× g for 10 min. Total proteins content was determined using a Pierce^TM BCA Protein Assay Kit (Thermo Fisher, San Diego, CA, USA). Subsequently, proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes (Biosharp, Beijing, China). The membranes were blocked with 5% nonfat milk for 2 h at room temperature. The blocked membranes were then incubated with the primary antibodies recognizing TLR4 (Proteintech, Rosemont, IL, USA), MyD88 (Cell Signaling Technology, Danvers, MA, USA), p65, p-p65, IκBα, p-IκBα, iNOS and COX-2 (Abcam, Cambridge, UK), The reaction was allowed to proceed, at 4 °C for overnight, after which the membranes were incubated with secondary antibodies for 1.5 h at room temperature. The immunoreactive proteins on the blots were detected using an enhanced chemiluminescence detection system (Tanon, Shanghai, China). β-actin (Cell Signaling Technology, Danvers, MA, USA) was used as the internal control, and band intensities were quantified using Image-J.

The total protein was lysed using the Pierce^TM RIPA Buffer (Thermo Scientific, 89900, Rockford, IL, USA) mixed with Halt^TM Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, 78442, Rockford, IL, USA). The quantitation of proteins was carried out using a Pierce^TM BCA Protein Assay Kit (Thermo scientific, 23227, Rockford, IL, USA). Protein samples were prepared by mixing an equal amount of protein with LDS Sample Buffer (Novex^TM by life technologies, B0008, Carlsbad, CA, USA) and Sample Reducing Agent (Novex^TM by life technologies, B0009) before denaturation. Gel electrophoresis was performed using a Mini Gel Tank chamber system (Invitrogen) with Bolt^TM 4–12% Bis-Tris gel (Invitrogen, NW04122BOX, Carlsbad, CA, USA), followed by transferring the protein to the Nitrocellulose Blotting membrane (Amersham^TM, 10600003, Germany). 5% BSA or nonfat milk was used to block nonspecific antigens, and primary antibodies were incubated overnight at 4 °C, including phospho-p44/42 ERK1/2 (CST, 4370, RRID: AB_2315112), p44/42 ERK (CST, 4695, RRID: AB_390779) and GAPDH (CST, 5174, RRID: AB_10622025). HRP-linked Anti Rabbit IgG (CST, 7074, RRID: AB_2099233) was used as secondary antibody. Examination of the signal was performed using Amersham Imager 600 (Pittsburgh, PA, USA) with SignalFire™ ECL Reagent (CST, 6883S, Frankfurt, Germany).

The cells were lysed in Pierce^TM RIPA Buffer (ThermoScientific, Waltham, MA, USA). The protein concentrations were established using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Western blotting was performed using standard procedures, and the blots were developed using Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA). Original images of western blot added to the Supplementary Data. Primary antibodies used were as follows: ERCC4 (#13465), p-ATM (Ser1981) (#13050), p-ATR (Ser428) (#2853), p-Chk1 (Ser345) (#2341), p-Chk2 (Thr68) (#2197), p-CDC25C (Ser216) (#9528), p-H2AX (Ser139) (#3257000) (1:1000; Cell Signaling Technology, Danvers, MA, USA); NEDD9 (ab18056, 1:1000), beta-Actin (ab49900, 1:100,000) (Abcam; Cambridge, MA, USA), Vinculin (700062) (1:1000, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Vinculin and beta-Actin were used as the loading controls. The quantification was performed using NIH ImageJ Imaging Software (Rayne Rasband, National Institutes of Health, USA).