Sod1 mouse 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.

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.