Loricrin

Extracts from different tissues (heart, brain and skin) were prepared from 3-day-old mice. Tissues were lysed with Ultra Turrax in boiling SDS Lysis Buffer (2% SDS, Tris HCl pH 7.5, 0.5 M EDTA), with protease inhibitor cocktail (Roche), and centrifuged at 13000 rpm for 15 min. Primary keratinocytes lysates were prepared by scraping 6-well dishes into 100 μl boiling SDS Lysis Buffer, vortexed and boiled several times. Lysates were subjected to western blotting analysis with the following antibodies: filaggrin, loricrin, keratin 1 and keratin 5 (Covance (Princeton, NJ, USA)); p130Cas, ΔNp63, E-cadherin, beta-catenin and alpha-catenin (BD Transduction Laboratories (San Jose, CA,USA)); tubulin and c-Src (Sigma (St. Louis, MI, USA)); phospho-Erk1/2 MAPKs (Thr202/Tyr204), phospho-Src (Tyr416), phospho-YAP and Yap (Cell Signaling (Danvers, MA, USA)); Erk1/2, CyclinD1, pTyr (Santa Cruz (Palo Alto, CA, USA)). Secondary antibodies were incubated for 1 h at RT, and detection performed with ECL Prime (GE Healthcare, Chicago, IL, USA). Protein band intensities were determined using the Image J software. All comparative images of blots shown are resulting from same exposures of the same membranes.

Paraffin-embedded mandible sections were dehydrated, rehydrated, and then pretreated in 10 mmol/L citrate buffer (pH 6.0, Sigma-Aldrich) for 20 min using a microwave for antigen retrieval. The specimens were blocked by Power Block (BioGenex) and incubated with primary antibodies against Med1 (Santa Cruz, TRAP220), Krt71 (Progen), and loricrin (Covance), Sox2 (Epitomics), Alpl (R&D system), Notch1 (Cell Signaling), and c-Notch1 (Cell Signaling). They were subsequently incubated with species specific secondary antibodies conjugated with fluorescent dyes (Invitrogen Molecular Probes), including Alexa 594 (red) and Alexa 488 (green). They were then counterstained with DAPI. The images were taken with a confocal microscope (LSM510, Carl Zeiss). Pictures were taken under magnification of 200.

For immunohistochemical and immunofluorescence stainings cryosections from Optimal Cutting Temperature compound (OCT, Tissue Tek) embedded tissues were fixed (4% PFA or in methanol), blocked (10% normal goat serum in PBS) and incubated with primary antibodies (diluted in blocking buffer) over night at 4 °C (ref. 60 (link)). Bound primary antibody was detected by incubation with peroxidase-conjugated (EnVision System, Dako) secondary antibody, followed by incubation with peroxidase substrate (Sigma), or Alexa-Fluor 488- or Alexa Fluor 594-conjugated antibodies (Invitrogen). Nuclei were counterstained with hematoxylin or 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). After washing slides were mounted in mounting medium. Images were taken with a Zeiss Meta 710 Confocal Microscope. Used primary antibodies and their dilutions (Supplementary Table 1): mTOR (Cell Signaling Technology, CST), S6-pS240/244 (CST); Akt-pS473 (CST); K14 (PROGEN Biotechnik); K10 (Covance); K14 (Covance); K15 (Covance); loricrin (Covance); filaggrin (Covance); p63 (Santa Cruz Biotech); P-cadherin (Zymed); active Caspase3 (CST); Survivin (CST), K8/18 (PROGEN Biotechnik); Par3 (EMD Millipore); LGN (EMD Millipore) and β4-integrin (BD Biosciences).

Slides were incubated with primary antibodies for 1 h at room temperature following rinsing with PBS. Antigen retrieval was performed for 10 min in TEG buffer. Slides were washed in 50 mM NH₄Cl in PBS for 30 min and blocked by 1% BSA, 0.2% gelatine, 0.05% Saponin in PBS at room temperature for 10 min, three times. Primary antibody was diluted in 0.1% BSA, 0.3% Triton X-100 in PBS, overnight at 4°C. Antibodies against involucrin, loricrin, and filaggrin were purchased from Covance (Denver, PA). Rabbit polyclonal antibody PAR2 (H-99; sc-5597) was provided by Santa Cruz Biotechnology (Dallas, TX). Slides were rinsed three times for 10 min in PBS containing 0.1% BSA, 0.2% gelatine, and 0.05% saponin at room temperature and the secondary biotinylated antibody (goat-anti rabbit; Vector Labs, Burlingame, CA) was diluted in 0.1% BSA, 0.3% Triton X-100 in PBS. Elite Standard Vectastain ABC kit and DAB kit (both Vector Labs) were finally applied according to the manufacturer's instructions. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Dako, Glostrup, Denmark).
For hematoxylin and eosin (HE)-staining, paraffin-embedded sections of 4 μm were used. Microscopic analyses were performed using an Axioskop2 (Zeiss, Oberkochen, Germany) microscope and the Axiovision software Rel4.7 (Zeiss).

Cell lysates were prepared by using the CelLytic M cell lysis reagent (Sigma-Aldrich). Protein levels were determined by using Micro-BCA Assay Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Ten micrograms of protein were loaded onto a NuPAGE Bis-Tris Gel. The blot was incubated with antibody against Notch1 (Cell Signaling), c-Notch (Cell Signaling), loricrin (Covance), and β-actin (Abcam), and subsequently with secondary antibody conjugated with HRP. The signals were visualized by an ECL kit (Thermo Scientific).

Mouse tissues were dissected and fixed in 10% buffered formalin or 70% ethanol and embedded in paraffin. Five μm-thick sections were used for H&E staining or immunohistochemical preparations. Antibodies used in immunostaining were: antibodies against HA (3724, Cell Signaling Technology); CYLD (SAB4200061), Involucrin (I9018) and Sma (C-6198) (Sigma-Aldrich); K5, K10, Filaggrin and Loricrin (Covance).