T per tissue protein extract reagent

Cells cultured to approximately 80% confluency in 6-well plates were harvested by trypsinization. Extraction of total RNA was performed with the TRIzol^® Reagents extraction kit (Thermo Fisher, San Jose, CA, USA). Synthesis of cDNA was performed using the Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher). Real-time PCR was performed using the MasterCycler RealPlex 4 from Eppendorf (Hamburg, Germany). The SYBR Green Master Mix was used for PCR reaction. Quantification of mRNA level was using the 2^-ΔΔT method.
For western blot, cells were lysed with T-PER Tissue Protein Extract Reagent (Thermo Fisher Scientific), supplemented with Proteinase Inhibitor Cocktail (Invitrogen) and phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich, Saint Louis, MO). Protein lysates of 30 μg was loaded into Mini-PROTEAN^® Precast Gel (4-20%), which was used for SDS-PAGE using the Tetra Vertical Electrophoresis Cell (Biorad). Resolved proteins were transferred to nitrocellulose membranes and incubated with antibodies according to standard procedures. Antibodies used in this study included: rabbit anit-TCF21 (ab32981, Abcam), rabbit anti-GAPDH (ab37168, Abcam), and HRP-conjugated goat anti-rabbit (ab6721, Abcam).

Tissues from multiple animals where pooled and homogenized with a polytron in 1:20 (w/v) of tissue to T-PER (Tissue-Protein Extract Reagent), pH 7.6 (ThermoFischer Scientific), in the presence of a protease inhibitor cocktail that inhibits serine, cysteine, aspartic and metalloproteases (Sigma-Aldrich, St Louis, MO, U.S.A). According to manufacturer instructions, tissue debris was pelleted at 10,000g for 5 minutes at 4°C and the lysate collected. For HAEC protein extraction, cells in culture flasks were washed three times in PBS and removed by scraping. Cells were collected by centrifugation at 200g for 5 minutes. T-PER was added to the cell pellet and was gently shaken for 10 minutes. The cell debris was removed by centrifugation 10,000g for 10 minutes at 4°C and the lysate collected. Protein concentrations were determined using the BCA protein assay kit (Pierce^TM, ThermoFischer Scientific) according to the manufacturer’s instructions. Protein lysates were stored frozen at -80oC until analyzed.