Gapdh rabbit mab

We used Western blotting to identify the presence of CYPs, antioxidants, and HPV proteins in Caski-derived EVs and to calculate the relative protein fold expression level of CYPs and AOEs in U1 cells upon treatment with CCS/CCS-EVs. We used the following primary antibodies: GAPDH Rabbit Mab, 1:2000 dilution (Cell Signaling Technology, Danvers, MA, USA), catalogue #2118; CYP1A1 rabbit Mab, 1:200 dilution (Proteintech Group, Inc., Rosemont, IL, USA), catalogue #13241-1-AP; CYP1B1 Rabbit Mab, 1:500 dilution (Santa Cruz Biotechnology, Dallas, TX, USA), catalogue #sc-32882; CYP2A6 Mouse Mab, 1:200 dilution (Abcam, Cambridge, MA, USA), catalogue #ab3570; SOD1 Mouse Mab, 1:1500 dilution, catalog #sc-101523; SOD2 Mouse Mab, 1:500 dilution, catalogue #sc-133254; Catalase Mouse Mab, 1:1200 dilution (Santa Cruz Biotechnology Inc., Dallas, TX, USA), catalog #21260-1-AP; PRDX6 Rabbit Mab, 1:500 dilution (LifeSpan Biosciences, Inc., Seattle, WA, USA), catalog #LS-C162131; CD63, Rabbit Pab, 1:200 dilution (Proteintech Group, Rosemont, IL, USA), catalog #25682-1-AP. CD81 rabbit Mab 1:400 dilution (Santa Cruz Biotechnology Inc., Dallas, TX, USA), catalog #sc-9158. While GAPDH was used as loading control for cellular proteins, CD63 and CD81 were used as loading controls for EV proteins.

Sodium selenite (Na₂SeO₃, Cat no: 214,485, ≥99% pure) and N-(1-pyrenyl) maleimide (NPM, Cat no: P7908, ≥98.5% pure) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-acetylcysteine amide (NACA, ≥99.9% pure) was provided by Dr. Glenn Goldstein (David Pharmaceuticals, New York, NY, USA). Casein zymogram gels (12% Ready Gel, Cat no: 161–1168) were purchased from Bio-Rad (Hercules, CA, USA). Rabbit m-calpain Antibody (Cat no: 2539S), GAPDH Rabbit mAb (Cat no: 2118S), and Anti-rabbit IgG, HRP-linked Antibody (Cat no: 7074S) were purchased from Cell Signaling Technology (Beverly, MA, USA). All other chemicals were purchased from Sigma-Aldrich, unless stated otherwise.

Purified protein samples were isolated using TRIzol reagent and subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the separated proteins were transferred onto PVDF membranes. The PVDF membranes were blocked for 1 h at room temperature and subsequently incubated with diluted primary antibodies at 4 °C overnight. After rinsing 3 times with TBST, the membranes were incubated with secondary antibody (goat anti-rabbit IgG-HRP, at 1:20000 in TBST; Maibio) for 1 h at room temperature. After rinsing, HRP was detected using the Millipore Immobilon Western Chemiluminescent HRP substrate, and the final blot was exposed to X-ray film. The bands were scanned and equilibrated to the protein concentrations of the samples. The following primary antibodies were used for western blotting: GAPDH rabbit mAb (Cell Signaling Technology, Cat No. 2118S), WNT4 rabbit mAb (Sigma, HPA011397), DVL2 rabbit mAb, (Proteintech, 12037-1-AP), GSK3 rabbit mAb (Cell Signaling Technology, 9369S), p-GSK3 rabbit mAb (Cell Signaling Technology, 9327S), and DNMT3A rabbit mAb (Abcam, ab23565).

Sodium valproate (VPA) and interleukin-2 was obtained from Sigma-Aldrich, St. Louis, MO, USA. Bovine serum albumin (BSA) and trypsin were purchased from Amresco, Solon, OH, USA. Fetal bovine serum (FBS), donor equine serum (DES), Alpha modified eagle medium (alpha-MEM), and Dulbecco’s modified eagle medium F12 (DMEM/F12) were obtained from Hyclone, Logan, UT, USA. Lapatinib, LY294002, rabbit polyclonal antibodies against PI3KCA, Akt Rabbit mAb, Phospho-Akt (Ser473) Rabbit mAb, HER3 Rabbit mAb, Phospho-HER3 Rabbit mAb, GAPDH Rabbit mAb, and goat anti-rabbit IgG antibodies conjugated to HRP were purchased from Cell Signaling Technology, Danvers, MA, USA. Anti-NKG2D mAb was obtained from R&D, Minneapolis, MN, USA. Phycoerythrin (PE)-labeled antibodies against human MICA and MICB and mouse IgG1 isotype control antibody were obtained from Biolegend, San Diego, CA, USA. Rabbit polyclonal antibodies against MICA and MICB were obtained from Santa Cruz, Santa Cruz, CA, USA.

For the western blotting analysis, cells were lysed on ice for 30 min in a lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors (10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 10 mg/ml aprotinin). Protein fractions were collected by centrifugation at 15 000 g at 4 °C for 10 min and then subjected to 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA and incubated with specific antibodies overnight at 4 °C. A horseradish peroxidase–labeled secondary antibody was added and visualized using an enhanced chemiluminescence detection system (Millipore, Billerica, MA, USA) as recommended by the manufacturer. We used the following primary antibodies to determine the concentrations of proteins in the lysates: anti-human RUNX2 rabbit mAb, anti-JAG1, HEY1 mAb (1 : 1000, Abcam, Cambridge, UK), anti-NOTCH1, HES1 mAb (1 : 1000, Cell Signaling Technology, Inc., Danvers, MA, USA), GAPDH rabbit mAb (1 : 1000, Cell Signaling Technology, Inc.), anti-HDAC1, HDAC2, HDAC3, HDAC5 mAb (1 : 1000, Abcam), anti-COL1a1, OCN (1 : 500, Abcam), and anti-AcH3 antibody (1 : 1000, Millipore, Bedford, MA, USA)).

To determine the expression of proteins of interest, 30 μg of proteins in 5% SDS were separated on a polyacrylamide gel (4% stacking, 10% resolving gel) at 150 V for 70 minutes. The proteins from the gel were transferred to a polyvinylidene fluoride membrane at 0.35 Amp for 90 minutes. The transferred blots were blocked with 5–10 mL of Li-Cor blocking buffer (LI-COR Biosciences, Lincon, NE) for 1 hour and incubated overnight with primary antibodies (GAPDH Rabbit Mab, 1:2000 dilution, Cell Signaling Technology, Danvers, MA; CYP1A1 rabbit Mab, 1:200 dilution, Abcam, Cambridge, MA; CYP3A4 Mouse Mab. 1:200 dilution, Santa Cruz Biotechnology. Inc. Dallas, TX; SOD1 Mouse Mab, 1:1500 dilution, Santa Cruz Biotechnology. Inc. Dallas, TX; Catalase Mouse Mab, 1:1200 dilution, Santa Cruz Biotechnology. Inc. Dallas, TX) at 4°C. After subsequent washing, the blots were incubated with corresponding secondary antibodies (1:10000 dilution, Goat anti-Mouse Mab, LI-COR Biosciences, Lincon, NE; 1:10000 dilution, Goat anti-Rabbit Mab, LI-COR Biosciences, Lincon, NE) for 1 hour at room temperature. The blots were scanned with Li-Cor Scanner (LI-COR Biosciences, Lincon, NE) and the densitometry data obtained from Image Studio Lite version 4.0 were used to calculate the fold expression of the proteins. GAPDH was used as an internal loading control to normalize the expression of sample proteins.