4–15 or 7.5% SDS–PAGE Mini-PROTEAN TGX gels (BioRad) were blotted using the semidry Trans-Blot Turbo Transfer system (BioRad).The following antibodies were prepared in 5% milk in TBS-T and incubated overnight at 4 °C: anti-Myc (1:1000, clone 4A6, Millipore), anti-p63 (1:2500, ab124762, Abcam), anti-p63α XP (1:1000, D2K8X, Cell Signaling), anti-GAPDH (1:20,000, clone 6C5, Merck), anti-p53 (1:500, DO-1, Santa Cruz), anti-PARP (1:1000, 9542, Cell Signaling), anti-cleaved caspase3 (1:1000, 5A1E,Cell Signaling), and anti-vinculin (1:1000, clone 7F9, Santa Cruz). Secondary antibodies, used for detection, were also prepared in 5% milk in TBS-T and incubated for 1 h at RT (goat anti-mouse HRP, 1:5000, A9917, Sigma–Aldrich; goat anti-rabbit HRP, 1:2000, Jackson Immuno Research Europe Ltd., 111–035–144). Quantification of western blot signals was performed by using ImageJ.
D2k8x
Manufactured by Cell Signaling Technology
The D2K8X is a piece of laboratory equipment designed for cell culture applications. It provides a controlled environment for growing and maintaining cells. The core function of the D2K8X is to regulate temperature, humidity, and CO2 levels to support optimal cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Goat anti rabbit hrp, by Jackson ImmunoResearch (1 mentions)
Mini protean tgx sds page gels, by Bio-Rad (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Anti myc, by Merck Group (1 mentions)
Goat anti mouse hrp, by Merck Group (1 mentions)
Anti p63, by Abcam (1 mentions)
Ab124762, by Abcam (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Anti vinculin, by Santa Cruz Biotechnology (1 mentions)
A9917, by Merck Group (1 mentions)
Anti p53, by Santa Cruz Biotechnology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
Topro, by Thermo Fisher Scientific (1 mentions)
Mist1, by Abcam (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Chip assay kit, by Merck Group (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
4 protocols using d2k8x
1
Western Blot Antibody Validation
2
Immunofluorescence Analysis of Submandibular Gland Tissue
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Paraffin embedded submandibular gland tissue sections were processed for immunofluorescence analysis as previously described [22 (link)]. Primary antibodies used at the indicated dilutions include ΔNp63 (1:50, Cell Signaling Technology, D2K8X), K5 (1:100, gift from Dr. Julie Segre), K14 (1:100 [45 ]), alpha-smooth muscle actin (Sma) (1:200, Sigma, 1A4), Aqp5 (1:100, Alomone Labs), K7 (1:50, Abcam), Mist1 (1:100, Abcam), Troma-III (K19, 1:50, Development Studies Hybridoma Bank), Ki67 (1:100, Leica Biosystems, MM1), and Cleaved Caspase-3 (1:100, Cell Signaling Technology, D175). Sections were stained with TOPRO (Invitrogen) and mounted using VECTASHIELD Antifade Mounting Medium (Vector Laboratories) and imaged using an Andor Dragonfly Spinning Disk Confocal Microscope with Fiji [46 (link)]. Microscopy data in this study was acquired at the Optical Imaging and Analysis Facility, School of Dental Medicine, State University of New York at Buffalo.
3
ChIP Assay for TP63 Binding Sites
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ChIP was performed using a ChIP assay kit (Millipore) according to the manufacturer's instructions. Briefly, cancer cells were incubated with 1% formaldehyde for 10 min at room temperature to crosslink their DNA. The cells were lysed in lysis buffer and sonicated to generate DNA fragments between 500 bp and 800 bp in length as assessed by agarose gel electrophoresis. Cross-linked chromatin was incubated at 4°C overnight with an antibody against TP63 (D2K8X, Cell Signaling Technology) or the isotype control (IgG). Before the IP, a sample of the input DNA was collected for normalization. After reversing the DNA–protein crosslinks, the immunoprecipitated DNA was purified using the QIAquick PCR purification kit (Qiagen) following the manufacturer's protocol. The final precipitated DNA was subjected to qPCR reactions using specific primers for the TP63 binding sites in the human miR-138-2 or TP21 (positive control [59 (link)]) promoters. The primers are listed in Supplementary Table 3 . The PCR result was normalized using the input DNA, and the specificity of the PCR amplification was confirmed by agarose gel electrophoresis.
4
Immunocytochemistry of HBE cells
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HBE cells on the transwell were fixed in 4% paraformaldehyde (PFA) for 30 min and subsequently washed with PBS. Cells were then permeabilized in 0.2% Triton X-100 in phosphate-buffered saline (PBST) for 10 min. Permeabilized cells were blocked in 10% non-specific goat serum and 1% bovine serum albumin (BSA) in PBST for 1 h. Primary antibody against p-H2AX (20E3, Cell Signaling Technology, Danvers, MA) was diluted at 1:200. Secondary antibody conjugated to Alexa Fluor 594 (ThermoFisher Scientific) was diluted at 1:500. Stained cells were mounted on coverslips and imaged with a fluorescent microscope (Axio Observer, Zeiss, Germany). To observe the differentiation of the ALI cultures, we stained fixed cells with primary antibodies for MUC5AC (45M1, ThermoFisher Scientific), FOXJ1 (2A5, ThermoFisher Scientific), β4-tubulin (ONS, Millipore Sigma), and p63α (D2K8X, Cell Signaling Technology). All primary antibodies were diluted at 1:500, followed by appropriate secondary antibody (AlexaFluor mouse/rabbit). Stained cells were counter stained for F-actin using phalloidin conjugated to AlexaFluor 488 (ThermoFisher Scientific) diluted at 1:40 and with Hoechst 33342 (ThermoFisher Scientific) diluted at 1:5000.
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