Trans blot turbo transfer pack 0.2 μm pvdf membrane

For western blot analysis, cells were lysed in RIPA buffer (MilliporeSigma) with protease inhibitors (MilliporeSigma) or isolated from TRIzol and dissolved in 1% SDS (Molecular Research Center, Inc.). Proteins were separated by SDS-PAGE, electroblotted onto Trans-Blot Turbo Transfer Pack 0.2 μm PVDF membrane (BioRad), and incubated overnight with antibodies, followed by corresponding secondary antibodies. Hikari signal enhancer kit was used to detect low abundance protein. For information about antibodies, see Key resources table.

For western blots, protein was collected using RIPA buffer (Life Technologies 89900), protease inhibitor (Thermo 1860932) and phosphatase inhibitor (Sigma 04906837001) and quantified using Qubit protein assay kit (Thermo Q33211). SDS-PAGE was run using 25–50 μg of protein mixed 1:1 with lamelli buffer (BioRad 161–0737) and heated to 90°C for 10 minutes and loaded onto a Mini-PROTEAN TGX gel (BioRad 456–8094). After gel electrophoresis, samples were transferred to TransBlot Turbo Transfer Pack 0.2 μm PVDF membrane (BioRad 1704156) using TransBlot Turbo (BioRad). Membrane was blocked using 5% BSA blocking solution for 30 minutes then primary antibodies were added at a 1:100 concentration overnight at 4C. The primary antibodies used were as follows: Anti-TEK (RD AF762), Anti-ANGPT2 (RD AF623), Anti-SMAD4 (Abcam ab40759), Anti-βACTIN (Cell Signaling 3700). After washing, appropriate LICOR IRDye 800CW or 680RD secondary antibodies were used at 1:1000 and incubated at room temperature for 1 hour. Membranes were imaged using Odyessy (LICOR) and iStudio Lite was used for western quantification.