P63α d2k8x

Immunostaining of tissue sections was performed as previously described [17 (link)]. IF was conducted using the antibodies p73 EP436Y (Abcam ab40658; 1:1000; Cambridge, UK) and p63α D2K8X (Cell Signaling Technology #13109; 1:1000; Danvers, MA), with TSA Plus Fluorescence Amplification Kit (Perkin Elmer, Waltham MA). Nuclei were counterstained with Slowfade Gold DAPI mounting media (Invitrogen, Carlsbad, CA). Micrographs of human tissues were all taken using a Zeiss slide scanner and further analyzed using Zeiss software. Micrographs of murine tissues were taken using a Leica microscope and camera and analyzed using Leica software; all scale bars = 50 µm.

Ovaries harvested from CD-1 PD5 mice were flash-frozen with liquid nitrogen at 0, 4, 8, 13, 18, 24, and 30 hours after CPA injection (150 mg/kg). The whole ovaries were homogenized in Pierce radioimmunoprecipitation assay buffer (89900, Thermo Fisher Scientific) with protease inhibitor (11836153001, Roche Diagnostics GmbH) and phosphatase inhibitor cocktails (04906837001, Roche Diagnostics GmbH). Proteins were loaded into the 4 to 15% Mini-PROTEAN TGX Precast Protein Gels (#4561084, Bio-Rad Laboratories Inc.) and transferred to a nitrocellulose membrane using the Trans-Blot Turbo Transfer System (#1704150, Bio-Rad Laboratories Inc.). The blots were probed with primary antibodies, followed by secondary antibodies. The catalog numbers and dilution of primary antibodies were as follows: p63-α (D2K8X) (13109s; 1:1000) and α/β-tubulin (2148s; 1:2000) from Cell Signaling Technology and DDX4 (ab270534; 1:2000) from Abcam. Proteins were detected using the Clarity Western ECL Substrate (#1705061, Bio-Rad Laboratories Inc.) and exposed using the iBright CL1500 Imaging System (A44114, Invitrogen).

Immunostaining of tissue sections was performed as previously described [3 (link)]. Murine skin tissues were fixed in 10% neutral buffered formalin (NBF) and embedded in paraffin for sectioning. De-identified human skin sections were obtained from pre-existing de-identified formalin-fixed paraffin-embedded tissue blocks. These blocks were prepared from excess tissue remaining after evaluation and diagnosis at the time of a surgical procedure. The Vanderbilt University Medical Center Institutional Review Board considers these tissues exempt since they were pre-existing and de-identified. IF was conducted using the following antibodies: p73 EP436Y (Abcam, ab40658, 1:1000), p63α D2K8X (Cell Signaling Technology, 13109, 1:1000), Keratin 5 (Fitzgerald Industries International, 20R-CP003, 1:200), Keratin 14 (Fitzgerald Industries International, 20R-CP002, 1:200), E-cadherin (BD Biosciences, 610181, 1:1000), Keratin 10 (Abcam, ab76318, 1:100), and Ki67 B56 (BD Biosciences, 550609, 1:1000). p73, p63, and Ki67 were detected using TSA Plus Fluorescence Amplification Kit (PerkinElmer). Keratins (5, 10, 14) and E-cadherin were detected using species-appropriate Alexa Fluor secondary antibodies at 1:200 (Thermo Fisher Scientific). IHC was conducted using γH2AX (Novus Biologicals, NB100-2280, 1:1000) antibody.