Mouse anti involucrin

Immunoblotting was conducted as described previously [34 (link)]. Briefly, cell lysates extracted using the Pierce M-PER Mammalian Cell Lysis Buffer supplemented with 1% SIGMAFAST^™ Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA) were denatured, separated on a 4–12% NuPage^® Novex^® Bis-Tris gradient gel (Life Technologies, Carlsbad, CA, USA) at 200 V for 45 min, and transferred onto a nitrocellulose membrane (LI-COR, Lincoln, NE, USA) at 30 V for 1 h. Proteins were detected by incubating first with primary antibodies (rabbit anti-HMOX-1 antibody, Cell Signaling, Danvers, MA, USA; mouse anti-acetylated α-tubulin, mouse anti-AKR1B10, rabbit-anti-DNAI1, and mouse anti-CDC20B, Sigma–Aldrich; mouse anti-CK13 and goat anti-MGMT, Santa Cruz, Dallas, TX, USA; mouse anti-involucrin, Thermo Fisher Scientific, Waltham, MA, USA; rabbit anti-ADH1C and rabbit anti-FANCD2, Abcam, Cambridge, MA, USA), followed by a 30-min incubation with the IRDye-conjugated secondary antibodies (LI-COR). Images were taken using an Odyssey^® CLx Imaging System (LI-COR). Densitometry was conducted using LI-COR Image Studio software.

Expression of key protein markers was measured at T1, T12, and PT20 using immunoblotting as described previously (Xiong et al. 2018 (link)). Briefly, proteins were denatured in a loading buffer containing 50 mM dithiothreitol (DTT), separated on a NuPage^® Novex^® 4–12% Bis–Tris gradient gel (Life Technologies, Carlsbad, CA), and transferred onto a nitrocellulose membrane (LI-COR, Lincoln, NE). Proteins were detected by incubating first with primary antibodies (rabbit anti-HMOX-1 antibody, Cell Signaling, Danvers, MA; mouse anti-acetylated α-tubulin, Sigma-Aldrich, St. Louis, MO; mouse anti-CK6, Santa Cruz, Dallas, TX; or mouse anti-involucrin, Thermo Fisher Scientific, Waltham, MA) and then IRDye-conjugated secondary antibodies (LI-COR). Images were taken using the LI-COR Odyssey^® CLx Imaging System and densitometry performed using the LI-COR Image Studio software.

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).