Anti cytokeratin 1

The cells were washed with 0.01M PBS and fixed in 4% formaldehyde for 30 min at room temperature. Cells were then incubated for 1 h with the following primary antibodies: rabbit polyclonal Bek (1:100 in PBS, Santa Cruz, CA), rabbit polyclonal Anti-Cytokeratin 1 (1:500, Abcam, Shanghai, China), rabbit monoclonal Anti-Cytokeratin 6 (1:100, Abcam), rabbit polyclonal Anti-Cytokeratin 16 (1:100, Abcam), and rabbit polyclonal Anti-PKC antibody (1:100, Abcam). The primary antibodies were visualized using goat anti-rabbit IgG-FITC (1:500 in PBS, Beyotime). Nuclei were stained with DAPI. Finally, the cover slips were mounted on the slides and fluorescence was visualized using a confocal laser fluorescence microscope (Carl Zesis Zen 2008, Carl Zeiss Inc., Germany). Photographic images were taken from five random fields.

The cells were lysed in 62.5 mMTris–HCl (pH 6.8) containing 2% SDS, and the protein concentration was determined using the Pierce BCA assay (Thermo Fisher Scientific, Rockford, IL). Mercaptoethanol and bromophenol blue were added to make the final composition equivalent to the Laemmli sample buffer. The samples were fractionated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto Immobilon-P membrane (Millipore, Billerica, MA,). Goat anti-rabbit HRP (1:5000, Beyotime) was used as secondary antibodies. Antibody binding was visualized via Pierce ECL reagents (Thermo Fisher Scientific, CA). We used rabbit polyclonal Bek (1:500, Santa Cruz, CA), rabbit polyclonal Anti-Cytokeratin 1 (1:1000, Abcam), rabbit monoclonal Anti-Cytokeratin 6 (1:500, Abcam), rabbit polyclonal Anti-Cytokeratin 16 (1:500, Abcam), rabbit polyclonal Anti-PKC antibody (1:500, Abcam) as primary antibodies, and monoclonal GAPDH antibody (1:1000, Beyotime) as controls. The quantification of protein bands was established using Band-Scan software (PROZYME, San Leandro®, California).

At 7 days after the completion of treatment, 4 mm punch skin biopsy specimens were taken from 20 rabbits. Skin biopsy specimens were fixed in 10% formalin, embedded in paraffin, and cut into 4 mm-thick sections. Sections were stained with hematoxylin-eosin or processed for immunohistochemical analysis. For the immunohistochemical staining, primary antibody rabbit polyclonal Bek (1:50), rabbit polyclonal Anti-PKC antibody (1:250), rabbit polyclonal Anti-Cytokeratin 1 (1:50, Abcam), rabbit monoclonal Anti-Cytokeratin 6 (1:20, Abcam), rabbit polyclonal Anti-Cytokeratin 16 (1:20, Abcam) were reacted for overnight at 4 °C, and secondary antibody goat anti-rabbit IgG-FITC (1:500 in PBS, Beyotime) was reacted for 1 hour at room temperature. Representative images were taken by confocal microscope.