Hklf4 af3640

Transdifferentiated and control fibroblasts grown on cover glasses were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min. Cells were then washed twice with PBS and permeabilized with 2.5% Triton X-100 in PBS for 10 min. After two washes, cells were blocked in 10% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Cells were then incubated at 4°C overnight with primary antibodies in 5% BSA in PBS with 0.1% Tween 20 (PBST). Primary antibodies used were as follows: Flag (M2, F1804, Sigma-Aldrich), hKLF4 (AF3640, R&D Systems), p63 (D2K8X, Cell Signaling Technology), and KRT14 (ab7800, Abcam). The next day, cells were washed 4 × 10 min with PBST, followed by incubation with Alexa Fluor–conjugated secondary antibodies (Life Technologies) in 5% BSA in tris-buffered saline with Tween 20 for 45 min at 37°C. Cells were then washed 3 × 10 min in PBS, incubated with 4′,6-diamidino-2-phenylindole (0.5 μg/ml) in PBS for 5 min, and washed twice with PBS. The slides were then mounted using VECTASHIELD antifade mounting medium (H-1000, Vector Laboratories) and incubated overnight in the dark at room temperature. The slides were then imaged with a Nikon Eclipse 80i fluorescent microscope.

Cells were lysed in radioimmunoprecipitation assay buffer containing 1% NP-40, 150 mM NaCl, 50 mM tris-Cl (pH 8.0), and 1% SDS supplemented with cOmplete, EDTA-free protease inhibitor (11873580001, Roche). Protein concentration was determined by bicinchoninic acid assay (BCA) protein assay (#23227, Life Technologies), after which equal amounts of proteins were loaded and separated by polyacrylamide gels. Proteins were then transferred to nitrocellulose membranes. Antibodies used in this study were as follows: Flag (M2, F1804, Sigma-Aldrich), hKLF4 (AF3640, R&D Systems), p63 (D2K8X, Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (10R-G109a, Fitzgerald), and KRT14 (ab7800, Abcam).