Filaggrin

Total protein was extracted from HaCaT cells and protein concentration was determined using Bradford protein assay kit (Bio-Rad Laboratories). 20 μg of protein extracts were separated by 10% SDS-PAGE and electrotransferred onto a polyvinyllidenedifluoride membrane. The membranes were incubated overnight at 4 °C with primary antibodies against IL-13Rα1 (1:1000; Abcam, Cambridge, UK), p-S6K1 (Thr389, 1:1000, Cell Signaling Technology, USA), S6K1 (1:1000, Cell Signaling Technology, USA), p-Akt (Ser473, 1:2000, Cell Signaling Technology, USA), Akt (1:1000; Abcam, Cambridge, UK), p-mTOR (Ser2448, 1:1000, Cell Signaling Technology, USA), mTOR (1:1000, Cell Signaling Technology, USA), filaggrin (1:1000; Abcam, Cambridge, UK), loricrin (1:200; Santa Cruz Biotechnologies, CA, USA), involucrin (1:1000; Abcam, Cambridge, MA, USA), and β-actin (1:1000; Abcam, Cambridge, UK). The secondary antibodies (Sigma-Aldrich, St. Louis, MO) were incubated in 5% skimmed milk powder. The SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc) was used to visualize the signals.

Primary antibodies reactive with mouse Sca-1, STAT3, TAK1, phospho-TAK1 (Thr184/187), phospho-STAT3 (Tyr705), and β-actin (Cell Signaling Technology, Danvers, MA), murine mast cell protease-1 (mMCPT1; BioLegend, San Diego, CA), filaggrin (Abcam, Cambridge, MA) and vaccinia antigens (Santa Cruz Biotech, Dallas, TX) were used according to manufacturer recommendations. Formalin-fixed, paraffin-embedded skin tissues were sectioned and processed as previously described [4 (link)]. Cell cytosolic fractionation and immunoblot of HEK-001 cell lysates were analyzed as previously described [4 (link)].

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).

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).