Loricrin

Collected cells were homogenized in RIPA lysis buffer (Atto, Tokyo, Japan) and then prepared by centrifuging at 10,000 g for 10 min. For nuclear and cytosolic fractions, cells were lysed with nuclear or cytoplasmic extraction reagents (Pierce/Thermo Scientific, Rockford, IL, USA) according to the manufacturer's protocol. Next, 30 μg of proteins was separated by 10–12% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking for 1 h at 37°C in 5% skim milk (in 1x TBS), the membranes were incubated with primary antibodies and then incubated with horseradish peroxidase-conjugated anti-IgG secondary antibody. Anti-IκB-α, P-IκB-α, NF-κB p65, STAT1, and P-STAT1 were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Antiinvolucrin, loricrin, and filaggrin were purchased from Abcam Inc. (Cambridge, MA, USA). An anti-β-actin antibody was purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). All bands were detected by enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA). The band intensities of specific proteins were quantified using Gelquant 2.7 (DNR Bio-Imaging Systems, Jerusalem, Israel).

Immunostaining was performed for the expression of keratinocytes specific markers: CK5, CK10, involucrin (Sigma Aldrich, USA), and loricrin (Abcam, UK) and dermal fibroblast specific markers, collagen-3 (Santa Cruz Biotechnology, USA), desmin (Santa Cruz Biotechnology, USA), FAP-α (Abcam, UK), and procollagen-1 (Santa Cruz Biotechnology, USA). Briefly, cells were washed with PBS three times and treated with 4% paraformaldehyde (PFA) for 15 minutes. The fixed cells were washed with PBS (5 × 3 times) and incubated with the primary antibodies overnight at 4°C. Incubation at room temperature with respective secondary antibodies (1 : 200) was performed for 1 hour at 37°C. After washing with PBS, cell nuclei were stained with DAPI (Sigma Aldrich, USA) and observed microscopically.

Mice skin tissue (dorsal and tail skin) at various postnatal day ages were collected and fixed by neutral buffered formalin (NBF) or directly embedded in OCT compound (Tissue-Tek) and frozen. Immunofluorescence assays (IF) and Immunohistochemical analysis (IHC-P) was performed as previously described^{7 (link)}. Paraffin embedded tissue blocks were sectioned and Haematoxylin and Eosin staining (H&E) was performed to analyze the phase of the hair follicle cycle. Immunofluorescence assays (IF) and Immunohistochemical analysis (IHC-P) was performed on OCT frozen tissue and Paraffin embedded block respectively²³ . Tail whole mount was performed as described previously^{48 (link)}. Briefly, tail skin was incubated in 5 mM EDTA followed by separation of epidermal sheet from dermis followed by fixing with 2% formaldehyde for 10 minutes. Nile red was used to stain the sebocytes of sebaceous gland and confocal microscopy was used for image acquisition. Primary antibodies used such as: CD34 (1:100, BD Pharmingen); α-6 integrin (1:100, BD Pharmingen); BrdU (1:250, Abcam); Ki67 (1:100; Novocastra); Filaggrin (1:1000, Abcam); Loricrin (1:1000, Abcam); K10 (1:1000, Abcam); Lrig1 (1:500, R&D systems) and Sox9 (1:500, Merck Millipore).

Native tissue and tissue models were fixed in Roti®-Histofix (Carl Roth, Karlsruhe, Germany) for 2 hours before generating histological cross-sections of 4 µm thickness. As an overview of general histological features, tissue slides were stained with hematoxylin and eosin (H&E; Morphisto, Frankfurt am Main, Germany). For immunohistochemical staining tissue slides were rehydrated and blocked with 5% donkey serum for 30 minutes. Subsequently, the primary antibodies cytokeratin 3/12 (1:100 dilution; Bioss, MA USA), cytokeratin 1 (1:1000 dilution; Abcam, Cambridge, United Kingdom), cytokeratin 14 (1:100 dilution; Sigma-Aldrich, Munich, Germany), involucrin (1:200 dilution; Thermo Scientific Fisher, MA USA) and loricrin (1:500 dilution; Abcam, Cambridge, United Kingdom) were added and incubated at 4 °C overnight. After washing, the slides were exposed to the matching secondary antibodies Alexa Fluor® 555 donkey anti-mouse or Alexa Fluor® 555 donkey anti-rabbit (1:400 dilution; Thermo Fisher Scientific, Waltham, MA USA) for 30 minutes at room temperature. The sections were covered with Fluoromount-G DAPI (Thermo Fisher Scientific, Waltham, MA USA) to visualize the cell nuclei.