Eblot l1 transfer system

BN-PAGE was performed as previously described with slight modifications (3 (link)). Three leaf disks (5 mm diameter) were homogenized with liquid nitrogen. 1× NativePAGE™ Sample Buffer (BN2008, Invitrogen™) with 1× protease inhibitor cocktail was added to the homogenized samples. The mixed samples were centrifuged at 20,000 g for 30 min at 4°C. Native PAGE™ 5% G-250 Sample Additive (BN2004, Invitrogen™) was added to the supernatant at a final concentration of 0.125%. Proteins were separated by Native PAGE™ Novex® 3 to 12% Bis–Tris Gels (BN1001, Invitrogen™) and transferred to polyvinylidene fluoride (PVDF) membrane using an eBlot™ L1 transfer system (GenScript). The target proteins were probed with corresponding antibodies.

Cells were harvested in chilled RIPA Cell Lysis Buffer with EDTA (GenDEPOT, Barker, TX, R4100‐010) with 1% protease and phosphatase inhibitor cocktails (Halt, ThermoFisher, Waltham, MA). After protein determination and normalization between conditions, SDS‐PAGE was performed using Tris‐HCl poured gels. Electrophoretic transfer from gel to nitrocellulose blot was performed with the eBlot L1 Transfer System (GenScript, Piscataway, NJ). Western blotting was performed using antibodies against YAP (Santa Cruz, Dallas, TX, clone H‐9, Cat. No. sc‐271134), phospho‐YAP (Ser127; Cell Signaling Technology, Danvers, MA, Cat. No. 4911), β‐actin (Santa Cruz, Dallas, TX, clone C4, Cat. No. sc‐47778), GAPDH (Cell Signaling Technology, Danvers, MA, 14C10, Rabbit mAb #2118), HA tag (Santa Cruz, Dallas, TX, clone F‐7, Cat. No. sc‐7392), and DYKDDDDK Tag (FLAG; Cell Signaling Technology, Danvers, MA, Cat. No. 2368S). After secondary HRP, Western Sure Chemiluminescent substrate (LI‐COR, Lincoln, NE) was applied. The LI‐COR C‐DiGit chemiluminescent blot scanner was used to scan blots and the Image Studio software (LI‐COR, Lincoln, NE) to quantify band intensities between conditions.

1 × 10⁶ Hela cells were seeded in six-well plates. Eighteen hours later, the cells were transfected with mRNA transcripts (10 μg/well) using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Forty-eight hours after transfection, the cells were washed with phosphate-buffered saline (PBS) pH 7.4 and lysed with RIPA lysis buffer (Thermo Fisher Scientific) for twenty minutes on ice. After centrifugation, the supernatant was collected and mixed with loading buffer with or without dithiothreitol and separated by 10% SDS-PAGE. The separated proteins transferred to polyvinylidene fluoride membranes was performed by an eBlot L1 transfer system (GenScript, China). The membrane was blocked with 5% non-fat milk in PBS buffer. Gn and Gc proteins were detected using anti-Gn (at a final concentration of 1 µg/mL) and anti-Gc monoclonal antibody (at a final concentration of 1 µg/mL), followed by secondary antibodies labeled with HRP (at a final concentration of 0.1 µg/mL, Abcam). The membranes were developed with a chemiluminescent substrate (Merck Millipore), and images were acquired with an iBright 1500 imaging system (Thermo Fisher Scientific).