Ezripa lysis kit

A 96-well plate was coated with a coating solution containing 0.6% sodium bicarbonate (Sigma-Aldrich) and 0.3% sodium carbonate (Sigma-Aldrich) in distilled water at 4 °C overnight. The plate was blocked for 4 h at room temperature using 5% skim milk. After washing with PBS containing 0.1% Tween 20 (TPBS), protein samples isolated using the EzRIPA lysis kit (TaKaRa) were added to the plate, which was incubated at 4 °C for 24 h. After washing with TPBS, the plate was incubated overnight with primary antibodies at 4 °C (Table S1). After the plate was washed, the plate was incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. After washing, the samples were incubated with 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich) for 15 min for color development. The reactions were stopped with an equal volume of 2 M H₂SO₄, and absorbance was measured at 450 nm using an ELISA plate reader (Multiskan SkyHigh Photometer; Thermo Fisher Scientific).

Cells and skin tissues were lysed using radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (EzRIPA lysis kit; TaKaRa, Tokyo, Japan). After sonication, samples were centrifuged at 14,000× g for 15 min at 4 °C. Protein concentration was determined using the bicinchoninic acid assay (Thermo Fisher Scientific). Equal amounts of protein were separated using 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes previously activated with methanol. PVDF membranes were blocked with 5% skim milk at room temperature to prevent the binding of non-specific proteins. Membranes were then incubated with appropriately diluted primary antibodies (Table S1) at 4 °C overnight. Membranes were rinsed with tris-buffered saline containing 0.1% Tween 20 and incubated with secondary antibodies at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence solution (Cytiva) using LAS-4000 (Bio-Rad, Hercules, CA, USA). Individual protein expression values were quantified using Image J software (National Institutes of Health, NIH, Maryland, MD, USA) and normalized to beta-actin (Cell Signaling Technology) to control the differences in protein loading. Values for a single blot are expressed relative to the mean of the first group.