PD-0332991 (palbociclib), a selective CDK4/6 inhibitor, was purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-phospho-H2AX (Ser139) antibodies were purchased from Millipore (Billerica, MA, USA). RB1, phospho-RB1 (Ser780; Ser807/811), E2F1, cyclin B1, cyclin D1, cyclin E1, CDK4, and CDK6 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin antibodies were purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-RAD51, cyclin A1, and CDKN2A antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cdkn2a
Manufactured by Santa Cruz Biotechnology
Sourced in United States
CDKN2A is a protein-coding gene that plays a role in regulating the cell cycle. It encodes two distinct proteins, p16INK4a and p14ARF, which have different functions in cell cycle control.
Automatically generated - may contain errors
Lab products found in correlation
Cyclin b1, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Cyclin a1, by Santa Cruz Biotechnology (1 mentions)
Anti phospho h2ax ser139 antibodies, by Merck Group (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Cyclin e1, by Cell Signaling Technology (1 mentions)
Anti rad51, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Abcam (1 mentions)
Benchmark ultra, by Roche (1 mentions)
Pik3ca, by Santa Cruz Biotechnology (1 mentions)
4 protocols using cdkn2a
1
CDK4/6 Inhibitor Palbociclib Protocol
2
Quantitative Western Blot and qRT-PCR Analysis
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Primary antibodies (RRAS2, 1:1000, cat. no. ab209078, Abcam; GAPDH, 1:2000–3000, cat. no. sc-32233, Santa Cruz Biotechnology; CDKN2A, 1:500–1000, cat. no. sc-1207, Santa Cruz Biotechnology) targeting proteins of interest were used for the Western blot. A detailed description is included in Supplemental Methods . Total RNA used for expression quantification was reverse-transcribed into cDNA using random primers, and then mRNA levels were quantified by qRT-PCR and normalized to that of GAPDH (Roche LightCycler). For pA site usage quantification, RNAs were reverse-transcribed with oligo(dT) primer, followed by PCR with two pairs of primers (proximal and distal) targeting different regions of the cDNAs. Specifically, the region targeted by the proximal pair is common to both APA isoforms and the region targeted by the distal pair is unique to the longer isoform. qPCR signals from the proximal and distal pairs of primers were compared to indicate the relative expression of the two isoforms. All primer sequence information is listed in Supplemental Table S9 .
3
Immunohistochemical Analysis of Cell Cycle and Receptor Proteins in Carcinoma and Pleomorphic Adenoma
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Carcinoma and pleomorphic adenoma samples underwent cell cycle and receptor protein expression analysis using a tissue microarray. Sections (approximately 2–3 µm) were used for automated immunohistochemistry (Benchmark ULTRA, Ventana Medical Systems, Tucson, AZ, USA) of CDKN2A (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TP53 (Dako, Glostrup, Denmark) and EGFR (Zytomed, Berlin, Germany).
Immunohistochemistry was scored semi-quantitatively as described previously17 (link): 0 (no apparent reaction), 1+ (positivity in <30% of tumour cells), 2+ (positivity in ≥30% but <60%) and 3+ (positivity in ≥60%). Immunostaining intensity was scored as 0 (absent), 1+ (weak), 2+ (intermediate) or 3+ (strong).
Immunohistochemistry was scored semi-quantitatively as described previously17 (link): 0 (no apparent reaction), 1+ (positivity in <30% of tumour cells), 2+ (positivity in ≥30% but <60%) and 3+ (positivity in ≥60%). Immunostaining intensity was scored as 0 (absent), 1+ (weak), 2+ (intermediate) or 3+ (strong).
4
Immunohistochemical Analysis of Tumor Markers
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The tumor tissues were fixed in 10% formalin for 6 hours and embedded in paraffin. Conventional slices (4 µm) were harvested on glass slides pre-treated with 2% (3-aminopropyltriethoxysilane) APES acetone and dried in a 60 °C oven for 1–2 hours. Immunohistochemistry staining was performed on the slices using the BenchMark XT automatic multi-function histopathological detection system for the following antibodies: CDKN2A (SC-1661, 1:200; Santa Cruz Biotechnology, Inc.), Calreticulin (CALR) (SC-373863, 1:200; Santa Cruz Biotechnology, Inc.), ROS Proto-Oncogene 1, Receptor Tyrosine Kinase (ROS1) (SC-376217, 1:100; Santa Cruz Biotechnology, Inc.), PIK3CA (SC-8010, 1:100; Santa Cruz Biotechnology, INC), Transcription Factor EB (TFEB) (SC-166736, 1:100; Santa Cruz Biotechnology, Inc.), TBXT (SC-166962, 1:50; Santa Cruz Biotechnology, Inc.), Chromodomain Helicase DNA Binding Protein 3 (CHD3) (SC-55606, 1:50; Santa Cruz Biotechnology, Inc.), Cut Like Homeobox 1 (CUX1) (SC-514008, 1:50; Santa Cruz Biotechnology, Inc.). The positive controls and negative controls of each antibody showed the appropriate results.
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