Protein Expression Analysis in HHF-SCs

Lysates of cultured HHF-SCs and keratinocytes were prepared by homogenization in a buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris (pH 8.0). Protein concentration was determined using Pierce BCA Protein Assay Kit (ThermoFisher Scientific Inc.). Subsequently, 25 µg of proteins were separated by SDS–8% PAGE, transferred onto a nitrocellulose membrane (Amersham Protran 0.45 NC), blocked with 5% milk solution in TBST, and subsequently incubated at 4 °C overnight with 1:250 rabbit anti-ABCA4 (cat. no. PA5-87983, Thermo Fisher Scientific Inc., as described previously [5 (link)]), 1:750 mouse anti-Involucrin (cat.no MA5-11803, Thermo Fisher Scientific Inc.), or 1:5000 mouse anti-β-actin (cat. no. A2228, Sigma-Aldrich, St. Louis, MO, USA). Secondary HRP-conjugated goat anti-rabbit IgG antibodies (cat. no. GTX213110-01, GeneTex, CA, USA) or rabbit anti-mouse IgG antibodies (cat. no. GTX213111-01, GeneTex) were used. The signal was detected with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific Inc.) and visualized with the use of the Syngene GBox Chemi XX9 System (Syngene, Cambridge, UK).