Bicinchoninic acid assay

Cells were lysed with a buffer containing 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, and 1% NP-40 supplemented with protease and phosphatase inhibitor cocktails (Roche). Cell debris were removed by centrifugation at 16,000g for 15 min (4°C). Thymus was smashed and homogenized in 1 ml of lysis buffer 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, and 1% NP-40 supplemented with protease and phosphatase inhibitor cocktails (Roche) using a TissueLyser (full speed for 20 min; Qiagen). The tissue was then maintained in constant agitation for 30 min at 4°C. Sample was centrifuged for 20 min at 13,000g at 4°C, and supernatant was collected. Both cell and thymus proteins were quantified using a bicinchoninic acid assay (Euroclone). Subsequently, 50 μg of cell lysates was run on an 8% polyacrylamide gel, and SDS-polyacrylamide gel electrophoresis was performed following standard procedures. After protein transfer, nitrocellulose membranes (Thermo Fisher Scientific) were incubated with anti-human/mouse ITPKB (ProteinTech), anti-human/mouse GAPDH [CST (Cell Signaling Technology), 14C10], or anti-human/mouse vinculin (E1E9V) antibody and developed using an enhanced chemiluminesence (ECL) substrate reagent (Thermo Fisher Scientific).

Pieces of lateral skin were cut, put in Eppendorf tubes, and immersed in liquid nitrogen for snap freezing. Tissues were smashed and homogenized in 1 ml of lysis buffer [50 mM tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, and 1% NP-40 supplemented with protease and phosphatase inhibitor cocktails; Roche] using a TissueLyser (full speed for 20 min, Qiagen). Samples were then maintained in constant agitation for 2 hours at 4°C and centrifuged for 20 min at 13,000g at 4°C. The supernatants were collected into a new Falcon tube. Proteins were quantified using a bicinchoninic acid assay (Euroclone). Cell lysates (150 μg) were run on a 10% polyacrylamide gel, and SDS–polyacrylamide gel electrophoresis was performed following standard procedures. After protein transfer, nitrocellulose membranes (Thermo Scientific) were incubated with the antibodies specific for phosphorylated SMAD2/3 (clone D27F4, Cell Signaling), mSerpin E1 (goat polyclonal IgG, R&D Systems), tPA (rabbit polyclonal, NOVUS Biologicals), and β-actin (rabbit polyclonal, Cell Signaling) and developed using an enhanced chemiluminesence substrate reagent (Thermo Scientific).