Journal: medRxiv

Article Title: Large tandem duplications in cancer result from transcription and DNA replication collision

doi: 10.1101/2023.05.17.23290140

Figure Lengend Snippet: a and b , Replicated-strand bias in head-on and co-directional collision regions in primary and metastatic cancers. The testing cohorts, tumor types, sample sizes, sources of replication orientation, head-on and co-directional collision regions are annotated on the top of the plots. P values are calculated by Fisher’s exact test. The x-axis shows Bonferroni-adjusted P values. c , TP53 mutations positively associated with large TDs in the PCAWG, Hartwig, and POG570 cohorts. The x-axis denotes individual tumors. The red bars indicate tumors with protein-altering somatic mutations. Cohort names and sample sizes are annotated above the plots. P values are calculated by linear regression with mutation load as covariates. Benjamini-Hochberg procedure is used to derive false discovery rates (FDRs). d , SPOP and CDK12 mutations positively associated with large TDs in the PCAWG prostate, Hartwig prostate and PCAWG ovarian cancers. e , PIK3CA mutations negatively associated with large TDs in four independent breast cancer cohorts. f , ARID1A and PTEN mutations negatively associated with large TDs in the PCAWG and Hartwig uterus cancers. g , Kaplan-Meier survival plots for patients with at least 10 large TDs and no large TD in their tumors. P values are calculated by log rank test. Cohorts and TP53 mutation status are annotated on top of plots.

Article Snippet: Antibodies used included: CDK12 (Atlas #HPA008038, 1:1000), GAPDH (Cell Signaling, 1:2000) and Lamin B1 (Cell Signaling, 1:1000).

Techniques: Mutagenesis

Journal: medRxiv

Article Title: Large tandem duplications in cancer result from transcription and DNA replication collision

doi: 10.1101/2023.05.17.23290140

Figure Lengend Snippet: a , Western blot (WB) showing CDK12 in 22Rv1 control (Ctrl) and two CDK12 KO clones (KO1 and KO2). GAPDH is shown as the loading control. b , Slot blot in 22Rv1 and CDK12 KO cells showing S9.6 (R-loops). RNAse H1 addition is used as a control. Single strand (ss) DNA blot is shown as the loading control. c , Quantification of relative S9.6/ssDNA signal (n=8 independent experiments). d , Examples of confocal microscopy images in 22Rv1 Ctrl and CDK12 KO cells. Blue indicates nuclei (DAPI) and red indicates PLA signal. DRB is used as a negative control. e , Quantification of the mean PLA signal per image per nuclei in 22Rv1 Ctrl and CDK12 KO cells (n=30 fields in one representative experiment). Three independent experiments were performed. f , WB showing CDK12 in 22Rv1 Ctrl, CDK12 KO, and CDK12 KO re-expressing wildtype CDK12. GAPDH is shown as the loading control. g , Quantification of S9.6 slot blot showing S9.6/ssDNA signal in CDK12 KO re-expressing wildtype CDK12 cells (n=5 independent experiments). h , Quantification of the mean PLA signal per image per nuclei in CDK12 KO + CDK12 re-expressing cells. In c , e , g and h , P values are calculated using ANOVA and corrected for multiple tests. Boxplots show medians (top bounds of boxes), standard errors of the mean (error bars) and individual data points (black dots, squares, triangles, and diamonds).

Article Snippet: Antibodies used included: CDK12 (Atlas #HPA008038, 1:1000), GAPDH (Cell Signaling, 1:2000) and Lamin B1 (Cell Signaling, 1:1000).

Techniques: Western Blot, Control, Clone Assay, Dot Blot, Confocal Microscopy, Negative Control, Expressing

Journal: medRxiv

Article Title: Large tandem duplications in cancer result from transcription and DNA replication collision

doi: 10.1101/2023.05.17.23290140

Figure Lengend Snippet: a , Working model of CDK12 loss leading to increased R-loops, TRCs and large TDs. b , Drug sensitivity measured by area under the curve (AUC) in association with the number of large TDs in cancer cell lines. Smaller values of AUC indicate cells are more sensitive to the drug, whereas large values indicate cells are more resistant. P values are calculated by Kendall test. c and d , Phase contrast images ( c ) and growth curves ( d ) of control (Ctrl) 22Rv1 and CDK12 KO cells (KO1 and KO2) treated with DMSO or 1 μM of MK-1775 for 15 days. Phase contrast images shown with the confluency mask (orange) are from day 15. P values are calculated by Student’s t test at day 15. Error bars represent standard errors of the mean.

Article Snippet: Antibodies used included: CDK12 (Atlas #HPA008038, 1:1000), GAPDH (Cell Signaling, 1:2000) and Lamin B1 (Cell Signaling, 1:1000).

Techniques: Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Defining CDK12 as a tumor suppressor and therapeutic target in mouse models of tubo-ovarian high-grade serous carcinoma.

doi: 10.1073/pnas.2426909122

Figure Lengend Snippet: Fig. 1. Commonalities of CDK12 loss in prostate and ovarian cancer. (A) Enrichment plots of CDK12-loss signatures derived from mCRPC in TCGA ovarian cancer with CDK12 inactivation. Genes are ranked by fold-change in 13 CDK12 inactivated cases vs. 34 selected control samples (Materials and Methods). (B) Scatter plot showing negative correlation (Pearson, same below) between CDK12 protein expression and CDK12-loss signature (up-regulated) score in TCGA ovarian cancer; 125 samples with available protein data are shown. Cases with CDK12 mutation are circled with thick lines and labeled. (C) Bar plot of correlation between CDK12 mRNA level and CDK12-loss signature (up-regulated) score across TCGA cancer types. Significant correlation with P value < 0.0001 is indicated by *. (D) Scatter plot showing hallmark pathways associated with CDK12-loss are concordant between ovarian cancer (OV) and mCRPC (cases from the Michigan Center for Translational Pathology, MCTP). Pathways with significant correlation (P value < 0.0001) are circled and labeled. BRCA, Breast invasive carcinoma; BLCA, Bladder Urothelial Carcinoma, PCPG, Pheochromocytoma and Paraganglioma, SKCM, Skin Cutaneous Melanoma, KIRC, Kidney renal clear cell carcinoma, TGCT, Testicular Germ Cell Tumors, PRAD, Prostate adenocarcinoma, PAAD, Pancreatic adenocarcinoma, UCEC, Uterine Corpus Endometrial Carcinoma, LUAD, Lung adenocarcinoma, LIHC, Liver hepatocellular carcinoma, THCA, Thyroid carcinoma, LUSC, Lung squamous cell carcinoma, CESC, Cervical squamous cell carcinoma and endocervical adenocarcinoma, SARC, Sarcoma, KIRP, Kidney renal papillary cell carcinoma, OV, Ovarian serous cystadenocarcinoma, HNSC, Head and Neck squamous cell carcinoma, STAD, Stomach adenocarcinoma, COAD, Colon adenocarcinoma, ESCA, Esophageal carcinoma, LGG, Brain Lower Grade Glioma, THYM, Thymoma, GBM, Glioblastoma multiforme.

Article Snippet: The primary antibodies used for immunoblotting include CDK12 (cat. no. 26816- 1- AP, Proteintech), CDK13 (cat. no., ABE1860, EMD Millipore), NF1 (H- 12) (cat. no.14623S, CST), Rb1 (cat. no. ab181616, Abcam), p53 (cat. no. NCL- L- P53- CM5P, Leica), GAPDH (cat. no. 3683S, CST), vinculin (cat. no. V9131, Sigma), and α- tubulin (cat. no. ab184577, Abcam).

Techniques: Derivative Assay, Control, Expressing, Mutagenesis, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Defining CDK12 as a tumor suppressor and therapeutic target in mouse models of tubo-ovarian high-grade serous carcinoma.

doi: 10.1073/pnas.2426909122

Figure Lengend Snippet: Fig. 2. Development of an ovarian cancer model with CDK12 inactivation. (A) Generation of mouse ovarian cancer model with Cdk12 knockout in mice carrying inactivated Trp53, Rb1, and Nf1 alleles (m-sgPRN and m-sgPRN;Cdk12KO mice). (B) Representative images of ovarian tumors and lymphoma in these animals. (C) Summary of oviductal tumor phenotype in m-sgPRN vs. m-sgPRN;Cdk12KO mice followed to humane endpoints. Weeks post-tamoxifen indicate the time points at which mice reached humane endpoints. L, left; R, right oviduct. Other (nonoviductal) tumors found at necropsy included sarcoma (Sa) and lymphomas (Ly). STIC, serous tubal intraepithelial carcinoma; eHGSC, early high-grade serous carcinoma; HGSC, high-grade serous carcinoma; MMMT, malignant mixed Müllerian tumors. (D) Kaplan–Meier plots demonstrating survival of m-sgPRN and m-sgPRN;Cdk12KO mice. (E) Histological images of representative ovarian tumors from m-sgPRN and m-sgPRN;Cdk12KO mice at endpoint. (F) Protein expression of CDK12, CCNK, NF1, p53, RB1, and vinculin (loading control) in luciferase-expressing cell lines generated from m-sgPRN and m-sgPRN;Cdk12KO models. (G) Representative bioluminescence images of NSG animals 1 wk after being intraperitoneally implanted with cells from (E). 4210(Luc) cells were generated from m-sgPRN;Cdk12WT while 4891(Luc) and 7201(Luc) were generated from m-sgPRN;Cdk12KO cells. (H) Quantification of bioluminescence signals from (G). The signal intensity of bioluminescence represented the tumor burden. P values show statistical intensity difference between line of indicated color and blue line [4210(Luc)].

Article Snippet: The primary antibodies used for immunoblotting include CDK12 (cat. no. 26816- 1- AP, Proteintech), CDK13 (cat. no., ABE1860, EMD Millipore), NF1 (H- 12) (cat. no.14623S, CST), Rb1 (cat. no. ab181616, Abcam), p53 (cat. no. NCL- L- P53- CM5P, Leica), GAPDH (cat. no. 3683S, CST), vinculin (cat. no. V9131, Sigma), and α- tubulin (cat. no. ab184577, Abcam).

Techniques: Knock-Out, Expressing, Control, Luciferase, Generated

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Defining CDK12 as a tumor suppressor and therapeutic target in mouse models of tubo-ovarian high-grade serous carcinoma.

doi: 10.1073/pnas.2426909122

Figure Lengend Snippet: Fig. 3. Increased immune cell infiltration in ovarian cancer models with CDK12 inactivation. (A) IHC for immune cell types in tumors from Cre-lox-driven Trp53/ Rb1/ Nf1 knockout mice with and without concomitant Cdk12 ablation. (B) Quantification of immune cells in (A). Bar graph data indicate the number of each cell type per high power field (n = 6 images from 6 mice/group). Data are presented as means ± SEM. (C) Protein expression of CDK12, CDK13, p53, RB1, NF1, and GAPDH (loading control) in individual cell lines generated from ovarian tumors of these animals. Protein expression in Myc-CaP prostate cancer cells shown for reference. (D) Immunofluorescence images demonstrate DNA damage, as indicated by γH2AX staining, in 15973 (PRN;Cdk12WT); 6227 (PRN;Cdk12KO); and 6137J (PRN;Cdk12HET) cell lines. (Scale bar, 25 µm.). Quantification of γH2AX signal.

Article Snippet: The primary antibodies used for immunoblotting include CDK12 (cat. no. 26816- 1- AP, Proteintech), CDK13 (cat. no., ABE1860, EMD Millipore), NF1 (H- 12) (cat. no.14623S, CST), Rb1 (cat. no. ab181616, Abcam), p53 (cat. no. NCL- L- P53- CM5P, Leica), GAPDH (cat. no. 3683S, CST), vinculin (cat. no. V9131, Sigma), and α- tubulin (cat. no. ab184577, Abcam).

Techniques: Knock-Out, Expressing, Control, Generated, Immunofluorescence, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Defining CDK12 as a tumor suppressor and therapeutic target in mouse models of tubo-ovarian high-grade serous carcinoma.

doi: 10.1073/pnas.2426909122

Figure Lengend Snippet: Fig. 5. Development of subcutaneous or intraperitoneal syngeneic allograft models of CDK12 loss ovarian cancer. (A) Generation of syngeneic allograft systems from cell line 6137J PRN;Cdk12HET generation via tamoxifen-inducible Cre activation. Generation of 6137J cell line from oviducal tumor. Subsequent growth as subcutaneous allograft (Top) or intraperitoneal administration following luciferase construct incorporation (Bottom). (B) Kaplan–Meier plot indicating time to increased tumor size following initiation of indicated agents. 6137J cells (5 × 106) administered subcutaneously; and treatment started upon tumor reaching 100 mm2, n = 8-10 tumors/ group. (C) Treatment of syngeneic allograft-bearing mice with CDK12/13 degrader YJ1206. (D) YJ1206 treatment of subcutaneously implanted OVCAR8_sgCDK12 (CDK12_KO) tumors. (E) YJ1206 treatment of subcutaneously implanted OVCAR8_sgNT (CDK12_WT). (F) Bioluminescence imaging of luciferase-expressing 6137J cell line in an intraperitoneal carcinomatosis model treated with vehicle vs. YJ1206. Mice imaged once per week. (G) Quantification of bioluminescence signals from (F). Graphs indicate tumor volume over the treatment time course.

Article Snippet: The primary antibodies used for immunoblotting include CDK12 (cat. no. 26816- 1- AP, Proteintech), CDK13 (cat. no., ABE1860, EMD Millipore), NF1 (H- 12) (cat. no.14623S, CST), Rb1 (cat. no. ab181616, Abcam), p53 (cat. no. NCL- L- P53- CM5P, Leica), GAPDH (cat. no. 3683S, CST), vinculin (cat. no. V9131, Sigma), and α- tubulin (cat. no. ab184577, Abcam).

Techniques: Activation Assay, Luciferase, Construct, Imaging, Expressing

Journal: Molecular cell

Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.

doi: 10.1016/j.molcel.2025.04.012

Figure Lengend Snippet: Figure 1. CDK12 depends on its RS domain for full activity (A) Schematic representation of the in vitro kinase assay. Phosphatase, lambda protein phosphatase; PP, protein phosphatase (inhibitors). (B) Coomassie blue-stained SDS-PAGE gel of the components of the in vitro kinase assay, incubated for the indicated times in min. Red arrows indicate phosphorylation products (also in E). C, control with no ATP added. Protein sizes are shown in kDa throughout the figures. (C) Schematic representation of the domain organization of wild-type and truncated constructs used in this study. CTD, C-terminal domain; NTD, N-terminal domain; KiD, kinase domain; RS, arginine and serine-rich domain. (D) Coomassie blue-stained gel showing purified CDK12/cyclin K wild-type (WT) or truncated mutant forms as in (C). (E) Coomassie blue-stained SDS-PAGE of an in vitro kinase assay using CDK12/cyclin K WT or truncated forms as in (C) on full-length RPB1 for the indicated times in min. C, control with no ATP added. (F) Western blot (FLAG) of in vitro binding reaction, mixing GST-CTD with the indicated forms of CDK12/cyclin K, followed by binding to glutathione Sepharose, and stepwise washes with increasing NaCl concentrations. FT, flow-through.

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), GST (Abcam, ab19256), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), RTF1 (Bethyl, A300-179A), alpha Tubulin (ab52866, Abcam), ATR (sc-515173, Santa Cruz), FANCD2 (FI17, sc-20022, Santa Cruz) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4◦C.

Techniques: Activity Assay, In Vitro, Kinase Assay, Staining, SDS Page, Incubation, Phospho-proteomics, Control, Construct, Purification, Mutagenesis, Western Blot, Binding Assay

Journal: Molecular cell

Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.

doi: 10.1016/j.molcel.2025.04.012

Figure Lengend Snippet: Figure 3. PAF1C subunit CDC73 directly contacts and specifically activates CDK12 (A) Western blot of the FLAG IP from HEK293 cells expressing FLAG-HA-CDK12. In, input; Ctr, no FLAG-HA-CDK12 expressed; FLAG, FLAG-HA-CDK12 ex- pressed. (B) Schematic of PAF1C and tags used for purification from insect cells. (C) Western blot of the in vitro binding reaction, mixing PAF1C and the indicated forms of CDK12/cyclin K, followed by Strep IP. Ctr, no PAF1C added. (D) Western blot of an in vitro kinase assay using CDK12/cyclin K and RNAPII complemented with PAF1C for the indicated times. Ctr, no PAF1C added; C, no ATP added; THZ, 2.5 μM THZ531 added. NTD, N-terminal domain; S2+S5, Ser2P + Ser5P. Red arrows indicate phosphorylation products throughout the figure. (E) Quantification of an in vitro kinase assay using CDK12/cyclin K and RNAPII comparing the presence and absence of PAF1C in the reaction in (D). (n = 3 independent experiments). Signal is mean with error bars indicating standard error. a.u., arbitrary units. (F) Western blot of an in vitro kinase assay using CDK9/cyclin T1 and RNAPII complemented with PAF1C for the indicated times as in (D). (G) Western blot of an in vitro kinase assay using CDK12/cyclin K and RNAPII complemented with PAF1C WT or incomplete variants for 5 min. ΔATP: no ATP. (H) Western blot of an in vitro kinase assay using CDK12/cyclin K and RNAPII complemented with PAF1C or FLAG-CDC73 titrated in for 5 min. C, no ATP added; THZ, 2.5 μM THZ531 added. (I) Quantification of an in vitro binding assay as in (C). Mean signal from CDK12 (FLAG) was normalized to St-CTR9 (negative control) to show the specific binding (n = 3 independent experiments). Signal is mean, with errors bars indicating standard error. **p < 0.01 as determined using an unpaired t test.

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), GST (Abcam, ab19256), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), RTF1 (Bethyl, A300-179A), alpha Tubulin (ab52866, Abcam), ATR (sc-515173, Santa Cruz), FANCD2 (FI17, sc-20022, Santa Cruz) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4◦C.

Techniques: Western Blot, Expressing, Purification, In Vitro, Binding Assay, Kinase Assay, Phospho-proteomics, Negative Control

Journal: Molecular cell

Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.

doi: 10.1016/j.molcel.2025.04.012

Figure Lengend Snippet: Figure 4. CDC73-KIM interacts with both cyclin K and CDK12 and promotes the opening of the CDK12 T-loop (A) (Left) AlphaFold2 structural model of CDK12 kinase domain (purple-blue), cyclin K cyclin fold (yellow) and CDC73-cyclin-K-interacting motif (KIM; green). The CDK12 active site indicated. (Right) Zoom-in showing T893 in the CDK12 T-loop in direct contact with Y290 of the CDC73-KIM. (B) (Top) Schematic of CDC73 domain organization. NTD, N-terminal domain. (Bottom) Multiple sequence alignment of CDC73-KIM in humans (Homo sapiens), zebrafish (Danio rerio), fruit fly (Drosophila melanogaster), and the worm (Caenorhabditis elegans). Fully conserved residues among the listed sequences are shaded in red. KIM regions important for T-loop opening and the cyclin K interaction indicated. (C) Zoom-in on AF2 structural model of CDK12 (purple)/cyclin K (yellow)/CDC73-KIM (green and light blue) complex from (A), highlighting in light blue the fully conserved residues as determined in (B).

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), GST (Abcam, ab19256), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), RTF1 (Bethyl, A300-179A), alpha Tubulin (ab52866, Abcam), ATR (sc-515173, Santa Cruz), FANCD2 (FI17, sc-20022, Santa Cruz) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4◦C.

Techniques: Sequencing

Journal: Molecular cell

Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.

doi: 10.1016/j.molcel.2025.04.012

Figure Lengend Snippet: Figure 6. CDC73-mediated activation of CDK12/13 is essential for balanced gene expression (A) Volcano plot of significance and difference of spike-in and library normalized gene expression across cell lines indicated relative to siControl sample. Significantly upregulated (log2FC > 1 and adjusted p value < 0.05) and downregulated (log2FC < −1 and adjusted p value < 0.05) genes indicated in red and blue, respectively. The number of differentially expressed events are similarly indicated. Log2FC, log2 fold change. (B) Boxplot of log2FC of library and spike-in normalized nascent RNA read counts relative to the siControl condition, across gene intervals divided according to length. Long: > ∼100 kb, mid2: ∼30–100 kb, mid: ∼10–30 kb, short: < ∼1–10 kb. ****p < 0.0001, *p < 0.1, and ns: not significant as determined using an unpaired t test. (C) Metagene analysis of non-overlapping coding genes between 1 and 10 kb (n = 2,442) using library- and spike-in normalized nascent RNA read counts obtained from transient transcriptome chem sequencing (TTchem-seq). Line indicates mean signal of experiment performed in technical triplicate. Shaded area indicates standard error. Si73, siRNA targeting CDC73; CPM, counts per million; TSS, transcription start site; TES, transcription end site; kb, kilobases. (D) Representative images of nascent RNA read counts across GADD45B and HSPA1A, with the direction of transcription indicated by arrow. (E) Western blot of whole cell extracts of Flp-In T-Rex HEK293 cell lines with or without knockdown of CDC73 and induction of recombinant FLAG-CDC73 siRNA resistant WT and KIM mutant forms. ATR and FANCD2 blotted to show the loss of protein expression in the siCDC73 and the KIM mutants.

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), GST (Abcam, ab19256), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), RTF1 (Bethyl, A300-179A), alpha Tubulin (ab52866, Abcam), ATR (sc-515173, Santa Cruz), FANCD2 (FI17, sc-20022, Santa Cruz) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4◦C.

Techniques: Activation Assay, Gene Expression, Sequencing, Western Blot, Knockdown, Recombinant, Mutagenesis, Expressing

Journal: Molecular cell

Article Title: PAF1C-mediated activation of CDK12/13 kinase activity is critical for CTD phosphorylation and transcript elongation.

doi: 10.1016/j.molcel.2025.04.012

Figure Lengend Snippet: Figure 7. Working model for PAF1C-mediated CDK12 activation and generation of CTD marks during transcript elongation Left, RNAPII-associated PAF1C recruits CDK12/13. Middle, recruitment results in increased CTD Ser2 Ser5 di-phosphorylation and a stable, Ser2 Ser5 di-phosphorylated RNAPII elongation complex, capable of efficient transcript elongation across long genes. Right, toward the end of genes, phosphatases such as SSU72 may associate with RNAPII and de-phosphorylate Ser5, leaving Ser2 mono-phosphorylation. Please note that some of the CTD repeats may be pre- phosphorylated at serine 5 (by CDK7 or CDK9), or Ser2 Ser5 di-phosphorylated (by CDK9), already prior to CDK12/13 action. For simplicity, other components of the elongation complex, such as DSIF and SPT6, are not shown.

Article Snippet: The membranes were incubated with antibodies against Ser2 (3E8), Ser5 (3E10) (1:1000 dilution, gift from D. Eick), Ser2 + Ser5 di-phosphorylated CTD (D1G3K, Cell Signaling), RPB1 N-terminal domain (D8L4Y, Cell Signaling), CDK9 (C12F7, Cell Signaling), Cyclin K (A301-939A, Bethyl), Lamin A (ab26300, Abcam), FLAG (D6W5B, Cell Signaling), GST (Abcam, ab19256), CDK12 (A301-679A, CrkRS, Bethyl), CTR9 (A301-395A, Bethyl), PAF1 (A300-172A, Bethyl), RTF1 (Bethyl, A300-179A), alpha Tubulin (ab52866, Abcam), ATR (sc-515173, Santa Cruz), FANCD2 (FI17, sc-20022, Santa Cruz) and CDC73 (A300-170A, Parafibromin, Bethyl) (1:1,000) overnight at 4◦C.

Techniques: Activation Assay, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Cyclin-Dependent Kinase 12 Increases 3′ End Processing of Growth Factor-Induced c-FOS Transcripts

doi: 10.1128/mcb.01157-14

Figure Lengend Snippet: Figure 1. CDK12 associates with the Exon Junction Complex (EJC) and SR splicing 724

Article Snippet: Anti-CDK12 antibody (87011, 97 Novus Biologicals) was used for ChIP and two anti-CDK12 antibodies (87012, Novus 98 Biologicals and ab57311, Abcam) were used for western blotting and RNA 99 immunoprecipitation (RNA-IP).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Cyclin-Dependent Kinase 12 Increases 3′ End Processing of Growth Factor-Induced c-FOS Transcripts

doi: 10.1128/mcb.01157-14

Figure Lengend Snippet: Figure 2. Depletions of CDK12 and eIF4A3 inhibit c-FOS activation by EGF. 757

Article Snippet: Anti-CDK12 antibody (87011, 97 Novus Biologicals) was used for ChIP and two anti-CDK12 antibodies (87012, Novus 98 Biologicals and ab57311, Abcam) were used for western blotting and RNA 99 immunoprecipitation (RNA-IP).

Techniques: Activation Assay

Journal: Molecular and Cellular Biology

Article Title: Cyclin-Dependent Kinase 12 Increases 3′ End Processing of Growth Factor-Induced c-FOS Transcripts

doi: 10.1128/mcb.01157-14

Figure Lengend Snippet: Figure 3. CDK12 depletion decreases levels of Ser2P, CstF64 and CSPF73 at the c- 773

Article Snippet: Anti-CDK12 antibody (87011, 97 Novus Biologicals) was used for ChIP and two anti-CDK12 antibodies (87012, Novus 98 Biologicals and ab57311, Abcam) were used for western blotting and RNA 99 immunoprecipitation (RNA-IP).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Cyclin-Dependent Kinase 12 Increases 3′ End Processing of Growth Factor-Induced c-FOS Transcripts

doi: 10.1128/mcb.01157-14

Figure Lengend Snippet: Figure 4. CDK12 and eIF4A3 depletions reduce 3’ end processing of c-FOS transcripts. 806

Article Snippet: Anti-CDK12 antibody (87011, 97 Novus Biologicals) was used for ChIP and two anti-CDK12 antibodies (87012, Novus 98 Biologicals and ab57311, Abcam) were used for western blotting and RNA 99 immunoprecipitation (RNA-IP).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Cyclin-Dependent Kinase 12 Increases 3′ End Processing of Growth Factor-Induced c-FOS Transcripts

doi: 10.1128/mcb.01157-14

Figure Lengend Snippet: Figure 5. Depletion of eIF4A3 impairs CDK12 recruitment to the c-FOS gene. 833

Article Snippet: Anti-CDK12 antibody (87011, 97 Novus Biologicals) was used for ChIP and two anti-CDK12 antibodies (87012, Novus 98 Biologicals and ab57311, Abcam) were used for western blotting and RNA 99 immunoprecipitation (RNA-IP).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Cyclin-Dependent Kinase 12 Increases 3′ End Processing of Growth Factor-Induced c-FOS Transcripts

doi: 10.1128/mcb.01157-14

Figure Lengend Snippet: Figure 6. N-terminal RS domain in CDK12 interacts with SRSFs. 851

Article Snippet: Anti-CDK12 antibody (87011, 97 Novus Biologicals) was used for ChIP and two anti-CDK12 antibodies (87012, Novus 98 Biologicals and ab57311, Abcam) were used for western blotting and RNA 99 immunoprecipitation (RNA-IP).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Cyclin-Dependent Kinase 12 Increases 3′ End Processing of Growth Factor-Induced c-FOS Transcripts

doi: 10.1128/mcb.01157-14

Figure Lengend Snippet: Figure 7. Dominant negative CDK12 proteins. 882

Article Snippet: Anti-CDK12 antibody (87011, 97 Novus Biologicals) was used for ChIP and two anti-CDK12 antibodies (87012, Novus 98 Biologicals and ab57311, Abcam) were used for western blotting and RNA 99 immunoprecipitation (RNA-IP).

Techniques: Dominant Negative Mutation