Ab109725

Tissue sections were processed as above and were stained with antibodies specific for CK5 (rabbit, 1:200, ab75869, Abcam, Cambridge, UK) and CK17 (rabbit, 1:100, ab109725, Abcam) for 2 h at RT. Sections were blocked using HRP Blocking Reagent (Abcam) EnVision+/HRP Visualization (Agilent, Santa Clara, CA, USA) and DAB substrate kit (Agilent) were used to visualize staining. Sections were counterstained with hematoxylin and mounted with Permount Mounting Medium (Fisher Scientific, Waltham, MA, USA). Representative photographs were taken under a light microscope at ×20 magnification.

Cells were washed with PBS and lysed by RIPA lysis buffer (Beyotime, Shanghai, China) with 1 mM phenylmethyl sulfonylfluoride (PMSF) (Beyotime, Shanghai, China). Protein concentrations were then quantified using a BCA protein assay kit (Beyotime, Shanghai, China). Subsequently, the proteins were denatured at 100°C for 10 min, loaded and separated on 10% SDS-PAGE gels and blotted onto PVDF membranes. Tris-buffered saline that contained 5% nonfat powdered milk was used to block non-specific binding for 1 h at room temperature. The membranes were incubated at 4°C overnight with the following antibodies: rabbit monoclonal K17 (ab109725, Abcam, Cambridge, MA, UK), rabbit polyclonal cleaved Caspase3 (ab2302, Abcam, Cambridge, MA, UK), rabbit polyclonal CyclinD1 (26939-1-AP, ProteinTech Group, Inc., Wuhan, China) and rabbit polyclonal GAPDH (10494-1-AP, ProteinTech Group, Inc., Wuhan, China). After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. Finally, ECL reagents (Thermo Scientific, Rockford, IL, USA) were used to detect the protein signals.