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Keratinocytes were grown on glass coverslips, fixed with 3,8% formaldehyde in PBS for 10 minutes and permeabilized with cold methanol for 5 minutes or fixed with cold methanol for 10 minutes. Paraffin-embedded skin sections were prepared following convential procedures. Samples were incubated for 1 hour (cells) or overnight (sections) in a wet chamber with antibodies against keratin 10 (sc-23877), phospho-histone H3 (sc-8656-R), cyclin A (sc-56299), cyclin E (sc-198), αTubulin (SC-23948) (all from Santa Cruz, CA, USA), involucrin (I-9018, Sigma-Aldrich, Inc), keratin 1 (PRB-149P, Covance, Vienna, VA, USA) or Ki67 (ab16667, Abcam, Cambridge, UK). Then, primary antibodies were revealed with Alexa Fluor-conjugated goat anti rabbit or anti mouse antibodies (Jackson ImmunoResearch, PA, USA). Samples were then incubated with 0.2 µg/ml DAPI (Vector Lab, Burlingame, CA). Coverslips were mounted with Prolong Gold Antifade Reagent (ThermoFisher Scientific, Waltham, MA) and cells were visualized and photographed with a Zeiss fluorescent microscopy. Immunofluorescence staining was scored by counting 400 cells.

Preparation of lysates and immunoblot analyses were performed as described previously (5 (link)) using Tris lysis buffer (50 mM Tris–HCl pH 7.8, 150 mM NaCl, 1% IGEPAL CA-630) containing 20 mM NaF, 20 mM β-glycerophosphate, 0.3 mM Na-vanadate, 20 μg/ml RNase A, 20 μg/ml DNase and 1/300 protease inhibitor cocktail (P8340, Sigma–Aldrich) and phosphatase inhibitor cocktail #2 (P5726, Sigma–Aldrich). Antibodies used in this study were purchased from the following sources: rabbit anti-Cdk2 (M2 SC-163, Santa Cruz Biotechnology), mouse anti-actin (ab-3280, Abcam, Cambridge, MA, USA), rabbit anti-GFP (ab-290, Abcam), mouse anti-tubulin (Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-Cyclin A (SC-751, Santa Cruz Biotechnology) and mouse anti-cyclin E (SC-198, Santa Cruz Biotechnology). Secondary antibodies used for western blot analysis were goat anti-mouse (31430) and, goat anti-rabbit (31460, Thermo Scientific). mouse anti-tubulin hybridoma cell line (clone #12G10) was developed by J. Frankel and E.M. Nelson under the auspices of the NICHD and maintained by the Developmental Studies Hybridoma Bank.