Ripa cell lysis buffer

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.

The treated cells were washed twice with cold phosphate-buffered saline (PBS), and proteins were extracted using RIPA cell lysis buffer (#R4100, GenDEPOT, TX, USA) with proteinase inhibitor (#BPI001, Biomax, Seoul, Republic of Korea). The protein concentration was measured using the Pierce^TM BCA protein assay kit (#23225, Thermo Fisher Scientific), and 40 μg of each protein sample was heated at 98 °C for 5 min and then run through 8% SDS-PAGE gel. The protein samples were then transferred to a 0.22 μm PVDF membrane (#GE10600021, GE Healthcare Life Sciences, Solingen, Germany) and blocked with 5% non-fat milk in 0.1% TBS-T for 1 h. The blotted membrane was probed using specific antibodies against COX-2 (#sc-166475, Santa Cruz Biotechnology, Dallas, TX, USA) and iNOS (#13120s, Cell Signaling Technology, Danvers, MA, USA), 1:1000 dilution with 5% non-fat milk in 0.1% TBS-T at 4 °C overnight. This was followed by washing thrice with 0.1% TBS-T solution for 10 min and probing with secondary antibodies (goat anti-rabbit IgG [H + L] secondary antibody, HRP, #31460, 1:5000 dilution, Thermo Fisher Scientific) for 2 h at RT. The blots were developed using Pierce™ ECL Western blotting Substrate (#32106, Thermo Fisher Scientific), visualized using Alliance Q9 mini (Cambridge, UK), and quantified using ImageJ software 1.52a (National Institutes of Health, Bethesda, MD, USA).

HUVEC total proteins on the chip and 2.5D collagen bed were extracted using RIPA cell lysis buffer (1×, GenDepot, USA) supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific, USA). Nucleus and cytoplasmic fractionation were performed using NE‐PER nuclear and cytoplasmic extraction reagents (Thermo Scientific, USA) following the manufacturer's manual. The concentrations of extracted proteins were measured using the Bradford assay (Bio‐Rad Protein assay dye reagent concentrate, USA). Exactly 10 µg of protein was used for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), then transferred to NitroPure nitrocellulose transfer membrane (LC7033‐300, GenDepot, USA) for blotting. Primary antibodies and HRP‐conjugated secondary antibodies were used to label target proteins. Protein expression was detected using West‐Q Pico Dura ECL solution (W3653, GenDepot, USA) and membranes were imaged using iBright CL750 Imaging System (A44116, Invitrogen, USA).
Expressions of VE‐cadherin and F‐actin protein were measured using ImageJ. VE‐cadherin and F‐actin band signals were normalized to the loading control signal. For better comparison, the normalized values were calculated as fold changes relative to the iso‐osmotic control.