Journal: Journal of Clinical Investigation

Article Title: Involvement of activation-induced cytidine deaminase in skin cancer development

doi: 10.1172/jci81522

Figure Lengend Snippet: Figure 4. Molecular characterization of spontaneous SCC in K14-AID–Tg mice. (A) Codon distribution of Hras1 somatic mutations. Black circles indicate nonsynonymous mutations. The number above each black circle indicates the codon number. (B) Base substitution patterns identified in Hras1 in spon- taneous and TPA-induced SCC. (C) Codon distribution of Trp53 somatic mutations. Black and white circles and gray rectangles indicate nonsynonymous, synonymous, and frameshift mutations, respectively. Frequency of TP53 somatic mutations in human skin SCC from the IARC TP53 database juxtaposed for comparison with distributions and hot spots seen in K14-AID–Tg mice. The top 4 codon numbers of mutations in human skin SCC are shown. (D) Base substitution patterns and frequency of the nucleotide 5′-flanking C-to-T transition in Trp53 in spontaneously developed SCC. The number in the center of the pie chart is the sum of the C-to-T and G-to-A changes. (E–H) Correlations of transcript levels between the K14-AID transgene and Ccnd1, Egfr, Trp53, and Pten, respectively, in spontaneous SCC. Pearson’s correlation statistic was used to measure the extent of the relationships between the expression levels of 2 genes. In A–D, data points indicate the individual K14-AID–Tg mice listed in Supplemental Table 2. (I) IHC of spontaneous SCC developed in K14- AID–Tg mice. Scale bar: 20 μm.

Article Snippet: Formalin-fixed, paraffin-embedded (FFPE) mouse tissue sections were stained using an avidin/biotin system with rat monoclonal anti-mouse AID Ab MAID-2 (66); rabbit polyclonal anti-cyclin D1 Ab (C-20; Santa Cruz Biotechnology Inc.); rabbit polyclonal antiEGFR Ab (sc-1005; Santa Cruz Biotechnology Inc.); rabbit monoclonal anti–phospho-p44/42 MAPK Ab (D13.14.4E, Thr202/Tyr204; Cell Signaling Technology); rabbit monoclonal anti–phospho-AKT Ab (D9E, Ser473; Cell Signaling Technology); and mouse monoclonal anti-p16 Ab (F-12; Santa Cruz Biotechnology Inc.).

Techniques: Comparison, Expressing

Journal: Journal of Clinical Investigation

Article Title: Involvement of activation-induced cytidine deaminase in skin cancer development

doi: 10.1172/jci81522

Figure Lengend Snippet: Figure 8. Working model of skin carcinogen- esis mediated by inflammation-induced AID expression. LPS, dsRNA, TPA, and UVB from the outside of the epidermis, and inflammatory cytokines from immune cells and fibroblasts increase AID expression in keratinocytes. AID increases cyclin D1 and EGFR expression and introduces mutations in Trp53 and Hras1 genes to activate the MAPK pathway. Repetitive stimulation and chronic inflammation maintain upregulation of AID, causing an accumulation of mutations in Trp53 and Hras1 genes and thereby promoting malignant transformation. Upward arrow indicates upregulation.

Article Snippet: Formalin-fixed, paraffin-embedded (FFPE) mouse tissue sections were stained using an avidin/biotin system with rat monoclonal anti-mouse AID Ab MAID-2 (66); rabbit polyclonal anti-cyclin D1 Ab (C-20; Santa Cruz Biotechnology Inc.); rabbit polyclonal antiEGFR Ab (sc-1005; Santa Cruz Biotechnology Inc.); rabbit monoclonal anti–phospho-p44/42 MAPK Ab (D13.14.4E, Thr202/Tyr204; Cell Signaling Technology); rabbit monoclonal anti–phospho-AKT Ab (D9E, Ser473; Cell Signaling Technology); and mouse monoclonal anti-p16 Ab (F-12; Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Transformation Assay

Journal:

Article Title: Developmental regulators containing the I-mfa domain interact with T cyclins and Tat and modulate transcription

doi: 10.1016/j.jmb.2007.01.020

Figure Lengend Snippet: The I-mfa protein is a novel partner of P-TEFb. (a) Schematic representation of the HIC and I-mfa proteins and their truncations used in this study. (b) I-mfa and HIC interact with P-TEFb in vivo. COS cells were transfected with plasmids expressing the FLAG-tagged proteins shown in panel B, or plasmid expressing FLAG tag alone as control. Cell extracts were subjected to immunoprecipitation with anti-FLAG antibody followed by Western blots probed with antibody against cyclin T1 (top panel) or CDK9 (third panel). Protein expression levels were monitored by probing western blots containing 2% of the input cell extract with antibody against cyclin T1 (second panel), CDK9 (fourth panel) and the FLAG epitope (bottom panel).

Article Snippet: Antibodies Goat anti-cyclin T1 antibody (T-18), rabbit anti-cyclin T1 antibody (H-245), rabbit anti-CDK9 antibody (C-20), and rabbit anti-HA (antihemagglutinin) antibody (Y-11) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vivo, Transfection, Expressing, Plasmid Preparation, FLAG-tag, Control, Immunoprecipitation, Western Blot

Journal:

Article Title: Developmental regulators containing the I-mfa domain interact with T cyclins and Tat and modulate transcription

doi: 10.1016/j.jmb.2007.01.020

Figure Lengend Snippet: I-mfa and HIC are substrates of P-TEFb. (a) P-TEFb is pulled down from HeLa cell extracts by GST-HIC and GST-I-mfa. HeLa cell extracts were incubated with the indicated GST fusion proteins bound to glutathione beads and P-TEFb binding was detected using Western blots probed with antibody against cyclin T1 or CDK9. GST was used as control. (b) GST fusion proteins used in the kinase assay shown in panel C. Proteins were stained with Coomassie Brilliant Blue. (c) GST pull-down and kinase assay. Complexes bound to GST-HIC or GST-I-mfa and to their truncated GST fusion proteins (panels (a) and (b)) were tested as substrates in kinase assays. (d) Immunopurified P-TEFb complexes used to phosphorylate purified GST fusion proteins as shown in panel (f). Complexes were immunoprecipitated (IP) from HeLa cell extract with anti-CDK9 antibody, examined by Western blotting with antibody against cyclin T1 or CDK9 (left panel), and used in kinase assays with the CTD4 peptide13 (middle panel) or GST-HIC (right panel) as substrate. DRB was present at 50μM where indicated. (e) Staining of the affinity purified GST fusion proteins that were used as substrates in the kinase assay shown in panel (f). (f) Immunoprecipitated P-TEFb complexes and affinity-purified GST or GST fusion proteins (panels (d) and (e)) were subjected to kinase assays. The Control lane contained kinase but no substrate.

Article Snippet: Antibodies Goat anti-cyclin T1 antibody (T-18), rabbit anti-cyclin T1 antibody (H-245), rabbit anti-CDK9 antibody (C-20), and rabbit anti-HA (antihemagglutinin) antibody (Y-11) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Binding Assay, Western Blot, Control, Kinase Assay, Staining, Purification, Immunoprecipitation, Affinity Purification

Journal:

Article Title: Developmental regulators containing the I-mfa domain interact with T cyclins and Tat and modulate transcription

doi: 10.1016/j.jmb.2007.01.020

Figure Lengend Snippet: I-mfa and HIC bind to cyclin T2 at two sites, identifying the conserved K/R-rich motif (KRM) adjacent to the cyclin domain. (a) Schematic representation of cyclin T1, T2a and T2b. (b) Cyclin T2 bind I-mfa and HIC. GST-cyclin T1 (GST-T1), GST-cyclin T2 (GST-T2b) and the truncations indicated were used to pull down in vitro synthesized 35S-labeled I-mfa in the absence or presence of 50 μM ZnCl2 (top and second panels, respectively) or 35S-labeled HIC in the absence or presence of 50 μM ZnCl2 (third and bottom panels, respectively). (c) Residues 250–300 adjacent to the cyclin domains are required for HIC and I-mfa binding. GST-T1, GST-T2 and their C-terminal truncations were used to pull down in vitro synthesized 35S-labeled I-mfa (top panel) or HIC (bottom panel) as indicated. (d) The K/R-rich motif (KRM) is required for I-mfa and HIC binding. The amino acid sequences of the KRM (aa 251–270 for T1 and aa 250–269 for T2) are shown for cyclins T1 and T2 (boxed). Basic residues are shown in bold and sequence similarities are marked as in Figure 1(a). The dotted line marks the TRM in cyclin T1. Lines denote the alanine substitution mutants used in the pull-down experiment below. In vitro synthesized 35S-labeled I-mfa, HIC, Tat, or CDK9 were pulled down by the indicated wild type GST-T1 C-terminal truncations and GST-T1 (1-303) alanine substitution mutants. A stained gel containing the GST fusion proteins used in this assay is shown. (e) Multiple alignment of cyclin T from different species. The three invertebrates have a single cyclin T gene. Amino acid residues that are identical in the alignment are highlighted in black. Those that are highly conserved within the cyclin T1 family or cyclin T2 family are highlighted in dark grey or light grey, respectively. The data were obtained from Ensembl Genome Browser (http://www.ensembl.org/). The alignment was processed with Clustal W (1.82) multiple sequence alignment program (http://www.ebi.ac.uk/clustalw/).

Article Snippet: Antibodies Goat anti-cyclin T1 antibody (T-18), rabbit anti-cyclin T1 antibody (H-245), rabbit anti-CDK9 antibody (C-20), and rabbit anti-HA (antihemagglutinin) antibody (Y-11) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vitro, Synthesized, Labeling, Binding Assay, Sequencing, Staining

Journal: Cell reports

Article Title: The HDAC-Associated Sin3B Protein Represses DREAM Complex Targets and Cooperates with APC/C to Promote Quiescence

doi: 10.1016/j.celrep.2018.11.024

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Cyclin F (C-20) , Santa Cruz , sc-952; RRID: AB_2071212.

Techniques: Transduction, Purification, Recombinant, Protease Inhibitor, DC Protein Assay, Bicinchoninic Acid Protein Assay, Transfection, Knock-Out, Plasmid Preparation, Cloning, Software, CRISPR