Proteinsimple fluorchem q

Radio-Immunoprecipitation Assay Lysis buffer (EMD Millipore, Burlington, MA, USA) was used to lyse HCAECs. Equal amounts of protein were subjected to 9% SDS-PAGE for electrophoresis, followed by transfer onto a polyvinylidene difluoride membrane. After blocking with 5% blocking buffer, the membrane was immunoblotted with primary antibodies overnight at 4 °C. The membrane was washed with tris-buffered saline/Tween-20 (0.2%) and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibodies. The corresponding bands were examined by a chemiluminescent HRP substrate kit (EMD Millipore, Burlington, MA, USA), and chemiluminescence signals were assessed using Proteinsimple + Fluorchem Q (Alpha Innotech, San Leandro, CA, USA). Antibodies against N-cadherin (Cat 610921, 1:2000), and vimentin (Cat 550513, 1:2000), E-cadherin (Cat 610182, 1:2000) from BD Biosciences (Franklin Lakes, NJ, USA), alpha smooth muscle actin antibody (α-SMA, 1:1000, Cat ab5694, Abcam) and GAPDH (Cat mab374, 1:3000) from EMD Millipore (Burlington, MA, USA) and endothelial Nitric Oxide Synthase (eNOS, Cat 32027, 1:2000) and VE-cadherin (Cat 32027, 1:2000) from Cell Signaling (Danvers, MA, USA) were used. The densitometry of the bands was analyzed using Image J software version (https://imagej.net/WelcomeUSA, accessed on 15 January 2022).

PTECs were lysed in RIPA Lysis buffer (EMD Millipore, Burlington, MA, USA). Equal amounts of protein were subjected to 9–11% SDS-PAGE for electrophoresis, followed by transfer onto a PVDF membrane. The membrane was blocked with 5% blocking buffer and sequentially immunoblotted with each primary antibody overnight at 4 °C. The membrane was washed with TBS/Tween-20 (0.2%) and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody. The corresponding bands were detected using a chemiluminescent HRP substrate kit (EMD Millipore, Burlington, MA, USA). The chemiluminescence signal was analyzed using Proteinsimple + Fluorchem Q (Alpha Innotech, San Leandro, CA, USA). The densitometry of the bands was analyzed using Image J software version 1.52. p53 Antibody (Catalog #9282), p27 Antibody (Catalog #3686s), Skp2 Antibody (Catalog #4358), Cyclin E1 (HE12) Mouse mAb (Catalog #4129), and Cyclin E2 Antibody (Catalog #4132) were obtained from Cell Signaling (Danvers, MA, USA). GAPDH antibody (Catalog #MAB374) was obtained from EMD Millipore (Burlington, MA, USA).