Ab187027

Total protein samples were extracted from cell samples with RIPA buffer (CWBio, Beijing, China) and protein concentration was measured using BCA protein assay kit (Beyotime, Shanghai, China). Equal amount of protein sample was separated on 12% SDS-PAGE gels followed by transferred to nitrocellulose membranes. Then, the membranes were blocked in 5% non-fat milk dissolved in TBST solution and incubated with primary antibodies against p300 (Abcam, Cambridge, MA, USA; ab275378, 1:1000 diluted), RhoA (ab187027, 1:2000 diluted), ROCK (ab134181, 1:1000 diluted), NF-κB p65(ab207297, 1:1000 diluted), HDAC1 (ab109411, 1:4000 diluted), COL1A1 (ab270993, 1:3000 diluted), COL2A1 (ab34712, 1:3000 diluted), and GAPDH (ab8245, 1:5000 diluted) overnight at 4°C. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 2 h. The protein bands were visualized using an enhanced chemiluminescence reagent (Pierce Biotech, Inc., Rockford, IL, USA).

RIPA lysis buffer supplemented with phosphatase and proteinase inhibitor (Roche, Basel, Switzerland) was used for protein extraction. Protein from cell lysates or tissues was separated on SDS-PAGE gel and transferred onto PVDF membrane (Merck Millipore, # IPVH00010). Western blot analysis was then performed using primary antibodies against VEGF165 (1:1,000, CST, #50661), VEGFR2 (1:1,000, CST, #9698), phospho-VEGFR2 (1:1,000, Tyr1175, CST, #2478), phospho-STAT3 (1:1,000, Tyr705, Abcam, #ab267373), STAT3 (1:2,000, Abcam, #ab68153), ERK (1:3,000, Abcam, #ab184699), phospho-ERK (1:1,000, Thr202/204, CST, #4370), RhoA (1:5,000, Abcam, #ab187027), phosphor-AKT (1:1,000, ser473, CST, #4060), AKT (1:1,000, CST, #9272), Cyclin D1(1:200, Abcam, #ab16663) and RhoA (1:5,000, Abcam, #ab187027). Secondary antibodies were employed to visualize the immunoblots. Image J software was utilized to quantitatively analyze protein expression.

After reaching 80% density of endothelial cells in the umbilical vein, we administered a drug stimulation for 30 min and then detected the CN-Patch activation of Rho GTPase through a pull-down technique. The protein extraction and quantification were the same as mentioned above. Active RhoA and Active CDC42 were measured using a pull-down assay kit from Cytoskeleton (16118, ThermoFisher, USA). The concentration of the primary antibody as RhoA (catalog number: ab187027, clone: EPR18134, Abcam) was 1:1000, and the CDC42 (catalog number: ab187643, clone: EPR15620, Abcam) antibody concentration was 1:10000. Following overnight incubation at 4 °C, the blots were washed in TBST (WB20500, NCM, China) and then incubated with IRDye® 800CW secondary antibodies in fresh blocking buffer for 1 hour at room temperature. Afterward, the blots were washed again in TBST. The images of the immunoblots were quantified using Image J software 1.8.0, and the relative levels of the target proteins were normalized by β-actin (dilution of 1:50000, catalog number: AC026, clone: ARC51105-01, ABclonal).

Radio-Immunoprecipitation assay cell lysis buffer (Amresco Inc., Texus, USA) was used to collect total protein. In brief, single cell suspension was centrifuged at 800 g at 4 °C for 5 min with the supernatant discarded. Then the cells were ice-bated in a fivefold volume of lysis buffer for 10 min, and then centrifuged at 12,000 g at 4 °C for 10 min to collect the supernatant. The collected protein was run on SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore). Subsequently, the membranes were blocked with 5% skimmed milk and then incubated with the primary antibodies against RhoA (1:5000, ab187027), p-RhoA (1:1000, ab41435), ROCK2 (1:5000, ab71598) and β-actin (1:1000, ab8227) (Abcam Inc., Cambridge, MA, USA) at 4 °C for 16 h, and then with the secondary antibody (1:3000, ab205718) at room temperature for 2 h. The immunoblotting image were visualized using the Image J software (Version 1.8.0, National Institute of Health, USA). Three independent experiments were performed.

The following antibodies were used for immunoblotting: anti-LC3 (L8918, Sigma-Aldrich), anti-paxillin (ab32084, Abcam), anti-Atg5 (#12994, Cell Signaling), anti-Atg7 (A2856, Sigma-Aldrich), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ab181602, Abcam), anti-FAK-pY397 (44-624G, ThermoFisher Scientific), anti-FAK (610087, BD Transduction Laboratories), anti-Src-pY416 (#2101, Cell Signaling), anti-Src (#2109, Cell Signaling), anti-RhoA (ab187027, Abcam), and anti-β-actin (ab8227, Abcam).