Krt14

Mouse tissue (frozen whole skin and muscle samples) and 3D-keratinocytes were homogenized and sonicated in CelLytic MT Mammalian Tissue Lysis/Extraction Reagent (Sigma), and then centrifuged at 16,000g for 10 min at 4°C. The supernatants were harvested, and equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were then incubated with primary antibodies to KRT5 (Abcam, Cambridge, UK), KRT10 (Abcam), KRT14 (Santa Cruz Biotechnology, Dallas, TX), desmoglein 1 (DSG1; GeneTex, San Antonio, TX), DSG2 (Abcam), desmocollin 3 (DSC3; Santa Cruz Biotechnology), α-tubulin (Cell Signaling), Akt (Cell Signaling), phospho-Akt (Ser473) (Cell Signaling), p70S6K (Cell Signaling), and phospho-p70S6K (Thr389) (Cell Signaling). After washing the membranes with 0.1% Tween 20 in PBS, they were exposed to a horseradish peroxidase-linked anti-rabbit IgG antibody (Cell Signaling). Bands were visualized with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) and their intensities were quantified using the program Multi Gauge (Fujifilm, Tokyo, Japan).

Nuclear and cytoplasmic proteins were extracted according to the manufacturer's instructions (Sigma, #NXTRACT). Western blotting was performed as described in detail in a previous study [26 (link)]. Primary antibodies used in current study included anti-mouse Yap1 (Abcam, #ab56701), TAZ (Cell signaling, #4883), Histone H4 (Santa Cruz, #sc-25260), Hsp90 (Santa Cruz, #sc-8262), p-Smad2 (Santa Cruz, #sc-101801), Smad3 (Cell signaling, #9513), p-Smad3 (Santa Cruz, #sc-11769), Krt14 (Santa Cruz, #sc-43310), Krt18 (Santa Cruz, #sc-45406) and p63 (Abcam, #ab124762).

Cytospins were prepared with freshly dissociated cells. After spinning, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 1% Triton X-100 in PBS for 10 min. Cells were blocked with 10% of normal goat serum for 30 min at 37°C, and then incubated with a primary antibody against CD44 (1:100; Santa Cruz Biotechnology), CD47 (1:100, Abcam) and KRT14 (1:100, Santa Cruz Biotechnology) for 1 h in humid atmosphere. After rising, cells were incubated with secondary goat anti-mouse IgG-Alexa Fluor 594-conjugated (1:100, Invitrogen), and the nuclei were counterstained with Hoechst (5 μg/mL). Negative controls were prepared by omitting staining with the primary antibody. Images were acquired in a confocal microscopy Zeiss LSM710 system (Carl Zeiss AG) using a × 63 1.4 NA oil immersion lens. The quantification of fluorescence intensities was performed using the NIH ImageJ 1.47v analysis software. Regions were drawn around each fluorescent cells and in a region without fluorescent objects for background subtraction. The corrected total cell fluorescence (CTCF) was determined using the following formula: CTCF = integrated intensity - (area for the selected cell × mean background). Data is represented as the mean of fluorescence intensity (MFI).