anti cdk12 rabbit polyclonal antibody  (Danaher Inc)

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: Cdk12 ablation in the prostate epithelium induces neoplasia (A) Prostate epithelial Cdk12 ablation scheme. (B) H&E staining and CDK12 immunohistochemistry in representative prostate samples from 52-week-old Cdk12 pc−/− mice (pure C57 background) and WT controls. Left panel scale bars, 100 μm. Other scale bars, 50 μm. (C) Bar graphs indicate percent cross-sectional area occupied by histologically abnormal tissue. AP, anterior prostate; DP, dorsal prostate; VP, ventral prostate; LP, lateral prostate. ( n = 8/group). (D) Pathological scoring (Path score) of prostate from the same animals. Numerical scores assigned to normal tissue (0), hyperplasia (1), focal HGPIN (2), and AIP (3) (indicated by respective images). Scale bars, 50 μm. Bar graph shows percentage prostate cross-sectional area occupied by tissue of each path score (scores 2 and 3 added together). ( n = 7–8/group). Statistical analysis with Mann-Whitney test. (E) Immunofluorescent staining of cytokeratin-8 (K8) and p63. 52-week-old Cdk12 pc−/− image shows an area of focal HGPIN with expansion of p63(+) BCs. Scale bars in left (100 μm) and right images (50 μm). (F) Ki67 immunohistochemistry in Cdk12 pc−/− or WT mice. Scale bars, 50 μm. Bar graph indicates percentage Ki67(+) cells per high-powered field ( n = 9 images from 3 mice/group). Data represented as mean ± SEM. (G) Immunohistochemistry for immune cell markers, indicating T cell-predominant infiltrate surrounding lesions in Cdk12 pc−/− animals. Scale bars, 100 μm. Bar graph data indicate number of each cell type per high-powered field ( n = 3–5 images from 3 mice/group). Box indicates standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Student’s t test used in (C), (F), and (G). See also Figure S1 .

Article Snippet: Rabbit polyclonal anti-CDK12 , Proteintech , Cat# 26816-1-AP; RRID: AB_2880645.

Techniques: Staining, Immunohistochemistry, MANN-WHITNEY, Standard Deviation

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: Organoids derived from the Cdk12 pc−/− prostate are morphologically abnormal, with impaired basal-luminal segregation (A and B) Images of organoids derived from Pb-Cre ; Cdk12 f/f ; mT/mG prostate BCs (52-week time point). Tom indicates Td-tomato-expressing cells with wild-type Cdk12 ( Cdk12 WT ). GFP indicates GFP-expressing cells with Cdk12 ablation ( Cdk12 KO ). Scale bars, 500 μm in (A) and 250 μm in (B). (C) CDK12 immunoblot in Cdk12 WT vs. Cdk12 KO organoids. (Vinculin, loading control). (D) Cdk12 KO organoid morphology: H&E staining, and CDK12 immunohistochemistry. Scale bars 200 μm in top and 50 μm in bottom panels. (E) Immunofluorescence for cytokeratin-8 (K8) and p63 indicating basal-luminal disorganization in Cdk12 KO organoids. Scale bars, 200 μm. (F) Uniform Manifold Approximation and Projection (UMAP) of scRNA-seq from Cdk12 WT organoids ( n = 3). The five identified cell states progress from Basal_1, Basal_2, Basal_3, Lum_1, to Lum_2. (G) UMAP of scRNA-seq from Cdk12 KO organoids ( n = 3). (H) Cells from Cdk12 KO organoids ( n = 3) projected into the UMAP of Cdk12 WT . Pseudocolor indicates presence (yellow) or absence (purple) of Cdk12 transcript. (I) Distributions of different cell states in Cdk12 WT and Cdk12 KO organoids. The most differentiated (Lum_2) population is lost in Cdk12 KO organoids. (J) GSEA for human CDK12 -loss signature—shared down-regulated genes from human PCa with CDK12 inactivation and siCDK12 knockdown LNCaP cells (18)— in Cdk12 KO organoids. Heatmap of logFC ( Cdk12 KO vs. Cdk12 WT organoids) for genes in signature. Genes contributing to negative enrichment (leading edge) are labeled. See also Figure S2 .

Article Snippet: Rabbit polyclonal anti-CDK12 , Proteintech , Cat# 26816-1-AP; RRID: AB_2880645.

Techniques: Derivative Assay, Expressing, Western Blot, Control, Staining, Immunohistochemistry, Immunofluorescence, Knockdown, Labeling

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: Cdk12 and Trp53 inactivating alterations interact to promote PCa (A) Workflow for CRISPR library screening of Cdk12 -interacting genes. (B) Snake plot representing log 2 fold change of guide RNAs in sequenced tumor samples described in (A). ( n = 3/group in 2 unique experiments). (C) Immunohistochemistry for p53 (left panels) and γH2AX (right panels) in prostates of one-year-old WT and Cdk12 pc−/− mice. Scale bars, 50 μm. Bar graph indicates percent γH2AX(+) cells from average of 3–5 sections from each of 3 mice. Data represented as mean ± SEM. t test used for individual comparisons. (D) Protein expression of p53 and γH2AX in Cdk12 WT and Cdk12 KO organoids (GAPDH, loading control). (E) CDK12-p53 co-staining in Cdk12 WT and Cdk12 KO organoids. Scale bars, 50 μm. (F) CRISPR-mediated Trp53 ablation in Cdk12 WT and Cdk12 KO organoids. sgp53 indicates Trp53 -specific guide RNA. sgNT indicates control non-targeting guide RNA. (α-tubulin, loading control). (G) Relative expression (Rel Exp) levels of Trp53 and p53 target genes in samples described in (F). ( n = 3/group). Data represented as mean ± SEM. One-way AVOVA used for statistical comparisons. (H) Cell proliferation in organoids from groups indicated in (F) measured by CellTiter-Glo (CTG assay). ( n = 3–4/group). Data represented as mean ± SEM. Two-way ANOVA used for statistical comparisons. (I) Kaplan-Meier plots indicating tumor formation during 70 days post-implantation of Cdk12 WT and Cdk12 KO organoids with or without Trp53 ablation ( n = 10/group). (J) Immunohistochemistry of AR, p53, CDK12, and γH2AX in Cdk12 KO -sgp53 allografts. Scale bars, 50 μm. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also Figure S3 and Table S1 .

Article Snippet: Rabbit polyclonal anti-CDK12 , Proteintech , Cat# 26816-1-AP; RRID: AB_2880645.

Techniques: CRISPR, Library Screening, Immunohistochemistry, Expressing, Control, Staining, CTG Assay

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: Cdk12/Trp53 double knockout allografts exhibit lymphocytic immune responses and increased sensitivity to ICB therapy (A) Growth of Cdk12 KO -sgp53, Myc-CaP, and TRAMP-C2 allografts in immunocompetent wild-type mice. ( n = 10–15/group). (B) Immunohistochemistry of CDK12, T cell markers (CD3, CD4, CD8), and natural killer cell marker granzyme B in Cdk12 KO -sgp53 allografts, Myc-CaP allografts, and TRAMP-C2 allografts, and prostates of Pten pc−/− PCa mouse model. Scale bars, 50 μm. (C and D) Tumor growth curve and endpoint weights of Cdk12 KO -sgp53 (C) and TRAMP-C2 (D) allografts treated with anti-PD1/CTLA4 cocktail. ( n = 8–14/group). (E) Flow cytometry-based quantification of CD4(+) and CD8(+) T cells (total, IFNγ(+), granzyme B(+)) in Cdk12 KO -sgp53 and TRAMP-C2 allograft samples +/− treatment with anti-PD1/CTLA4 cocktail. ( n = 7–8/group). (F) Kaplan-Meier plots demonstrating survival of prostate-specific Pten pc−/− and Pten pc−/− Cdk12 pc−/− mice. (G) Genitourinary tract weights of Pten pc−/− and Pten pc−/− Cdk12 pc−/− mice as well as wild-type mice (52 weeks). (H) Cell proliferation in complete media of epithelial cell organoids derived from Pten pc−/− and Pten pc−/− Cdk12 pc−/− mice measured by CTG assay. ( n = 4/group). Data represented as mean ± SEM. Data represented as mean ± SEM. One-way ANOVA for multiple comparisons (G), two-way ANOVA for multiple variables (C) and (E), and unpaired t test was used for tumor weight in (C) and (D). ∗∗∗∗ p < 0.0001; ns, not significant. See also Figures S4 and .

Article Snippet: Rabbit polyclonal anti-CDK12 , Proteintech , Cat# 26816-1-AP; RRID: AB_2880645.

Techniques: Double Knockout, Immunohistochemistry, Marker, Flow Cytometry, Derivative Assay, CTG Assay

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: Cdk12 ablation increases AR- and MYC-mediated signaling and promotes TRCs (A) Protein expression of CDK12, AR, and FOXA1 in multiple monoclonal Cdk12 WT and Cdk12 KO organoid lines. (GAPDH, loading control). (B) Gene set enrichment of AR target genes (activated and repressed) in Cdk12 KO organoids compared to Cdk12 WT . (C) Proliferation of Cdk12 WT and Cdk12 KO organoids grown in the absence of epidermal growth factor (EGF) and dihydrotestosterone (DHT) as measured by the CTG assay. ( n = 3 replicates per group in 2 unique experiments). (D and E) Morphology and viability quantification of Cdk12 WT and Cdk12 KO organoids subjected to enzalutamide (Enza) treatment. ( n = 3 replicates per group in 2 unique experiments). ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant. (F) Protein expression of CDK12, MYC, BRD4, BRD3, and BRD2 in Cdk12 WT and Cdk12 KO organoid lines. (G) Gene set enrichment of MYC target genes in Cdk12 KO organoids compared to Cdk12 WT . (H) Morphology of Cdk12 WT and Cdk12 KO organoid lines treated with JQ1 (1 μM). ( n = 3/group in 2 unique experiments). (I) Viability curves and IC 50 values for JQ1-treated Cdk12 WT and Cdk12 KO organoid lines. (J) Dot blot analysis quantifying R-loops in Cdk12 WT and Cdk12 KO organoids. RNase H1 treatment serves as a negative control. (K) Immunofluorescence images of R-loop (red) staining of Cdk12 WT and Cdk12 KO organoids (left) and quantification of fluorescence intensity (right). 100–200 cells/group. (L) Experimental workflow for identification of TRCs. Briefly, 2.5 mM of Thymidine was used to synchronize the cells, and 75 μM of DRB was used to inhibit transcription. (M) Representative immunofluorescence images of γH2AX staining in organoids treated as described in (L). (N) Quantification of γH2AX-positive cells in (M); ( n = 6/group, 3 unique experiments conducted). (O) Representative immunofluorescence images of γH2AX staining in unsynchronized organoids. (P) Quantification of γH2AX-positive cells in (O); n = 6–8 per group (3 unique experiments conducted). (Q) Detection of TRC by PLA assay. (R) Quantification of PLA foci per nucleus in (Q); 100–400 cells analyzed per group (2 unique experiments conducted). Data represented as mean ± SEM. One-way ANOVA for multiple comparisons, two-way ANOVA for multiple variables.

Article Snippet: Rabbit polyclonal anti-CDK12 , Proteintech , Cat# 26816-1-AP; RRID: AB_2880645.

Techniques: Expressing, Control, CTG Assay, Dot Blot, Negative Control, Immunofluorescence, Staining, Fluorescence

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: Cdk12 KO organoids and CDK12 -mutant tumors are preferentially sensitive to a CDK13/12 degrader (A) Snake plot representing data from siRNA screen for CDK12 synthetic lethal effects via 1NM sensitivity in CDK12 as cells. Negative Z scores indicate CDK12 synthetic lethal effects, with CDK13 representing most profound effect. (B) Immunoblot indicating CDK13 gene silencing with two different siRNAs (siCDK13.1 and siCDK13.2). (C) Curve depicting cell survival in 1NM-exposed CDK12 as cells transfected with one of two unique CDK13 siRNAs (siCDK13.1 and siCDK13.2) or control siRNA (siCON). (D) CRISPR-mediated Cdk13 (sgCdk13(1 + 3), or sgCdk13(2 + 4)) knockout in Cdk12 WT and Cdk12 KO organoids harvested on day 5 after lentiviral transduction. Protein expression of CDK12 and CDK13 in organoids (Vinculin, loading control). (E) Bright-field images of organoids described in (D). Scale bars, 200 μm. (F) Relative growth quantification from images in (E). ( n = 3/group). (G) CRISPR ablation of Cdk12 (sgCdk12) and Cdk13 (sgCdk13(1 + 3), or sgCdk13(2 + 4)) in Myc-CaP cells. Protein expression of CDK12 and CDK13 in Myc-CaP cells treated with indicated sgRNAs. (H) Colony formation assay showing survival in cells treated with indicated sgRNAs (representative data from 3 unique experiments). (I) Relative growth quantification from images in (H) (analysis of 11 high-powered fields per sample over 2 unique experiments). (J) (Top panel) C4-2B cells subjected to CRISPR-based CDK12 ablation ( CDK12 KO) or control sgRNA (C4-2B CTRL): percent confluence with siRNA-based CDK13 knockdown (si CDK13 ) or control siRNA (siNTC). (Bottom panel) C4-2B CDK12 KO and C4-2B CTRL cells: percent confluence with siRNA-based CCNK knockdown or control siRNA treatment. ( n = 3/group). (K) Images of Cdk12 WT and Cdk12 KO organoids (with or without Trp53 ablation) following treatment with CDK12/13 degrader (YJ9069). sgp53 indicates Trp53 ablation, while sgNT indicates intact Trp53 . Scale bars, 1,000 μm. (L) Viability curves and IC 50 values for YJ9069 treatment of groups described in (K). ( n = 4) (M) Protein expression of p-Ser RNA Pol-II, CDK12, CDK13, and p53 in Cdk12 WT and Cdk12 KO organoids with or without Trp53 ablation subjected to YJ9069 degrader or vehicle treatment. (N) IC 50 of organoids derived from PDX lines with WT CDK12 (MDA153, MDA146-12, LuCaP23.1, LuCaP86.2, LuCaP96, PC295) and inactivating CDK12 mutation (LTL706B, MDA117, MDA328). ( n = 3 per line). Data represented as mean ± SEM. One-way ANOVA for multiple comparisons, two-way ANOVA for multiple variables, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. See also Figures S6 , and Table S2 .

Article Snippet: Rabbit polyclonal anti-CDK12 , Proteintech , Cat# 26816-1-AP; RRID: AB_2880645.

Techniques: Mutagenesis, Western Blot, Transfection, Control, CRISPR, Knock-Out, Transduction, Expressing, Colony Assay, Knockdown, Derivative Assay

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: CDK13/12 degrader inhibits CDK12 -mutant tumor growth in vivo (A) In vivo treatment of Cdk12 KO -sgp53 allografts with YJ9069 or vehicle: line graph indicates tumor volume normalized to baseline. Bar graph indicates tumor weight at endpoint. ( n = 9–10 mice, each with 2 tumors, per group) (B) In vivo treatment of TRAMP-C2 allografts with YJ9069 or vehicle: graphs as indicated in (A) ( n = 9–10 mice, each with 2 tumors, per group). (C and D) Unmodified (sgNT-treated) Myc-CaP allografts (C) or sgCdk12-treated Myc-CaP allografts (D) subjected to in vivo YJ9069 treatment: line graphs indicate tumor volume. Bar graphs indicate tumor weight at end of treatment time course. ( n = 6–9/group). (E–H) CDK12 immunohistochemistry and TUNEL staining of unmodified (sgNT-treated) Myc-CaP allografts (E) and sgCdk12-treated Myc-CaP allografts (F). Bar graphs (G and H) indicate quantification of TUNEL(+) cells per high-powered field (scale bars, 50 μm). (I and J) YJ9069 treatment of subcutaneously implanted PDX lines, LTL706B ( CDK12 -mutant), and MDA146-12 (intact CDK12 ). Graphs indicate tumor volume ( n = 8–9 mice, each with 2 tumors per group). Two-way ANOVA used for tumor volume in (A), (D), and (I); unpaired t test used for tumor weight in (A–D) and (I and J) and TUNEL staining (G and H). ∗∗∗∗ p < 0.0001; ns, not significant. See also Figure S7 .

Article Snippet: Rabbit polyclonal anti-CDK12 , Proteintech , Cat# 26816-1-AP; RRID: AB_2880645.

Techniques: Mutagenesis, In Vivo, Immunohistochemistry, TUNEL Assay, Staining

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CDK12 , Proteintech , Cat# 26816-1-AP; RRID: AB_2880645.

Techniques: Virus, Recombinant, Plasmid Preparation, Blocking Assay, Lysis, Protease Inhibitor, Membrane, Transfection, SYBR Green Assay, Cell Viability Assay, Avidin-Biotin Assay, Software

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: Cdk12 ablation in the prostate epithelium induces neoplasia (A) Prostate epithelial Cdk12 ablation scheme. (B) H&E staining and CDK12 immunohistochemistry in representative prostate samples from 52-week-old Cdk12 pc−/− mice (pure C57 background) and WT controls. Left panel scale bars, 100 μm. Other scale bars, 50 μm. (C) Bar graphs indicate percent cross-sectional area occupied by histologically abnormal tissue. AP, anterior prostate; DP, dorsal prostate; VP, ventral prostate; LP, lateral prostate. ( n = 8/group). (D) Pathological scoring (Path score) of prostate from the same animals. Numerical scores assigned to normal tissue (0), hyperplasia (1), focal HGPIN (2), and AIP (3) (indicated by respective images). Scale bars, 50 μm. Bar graph shows percentage prostate cross-sectional area occupied by tissue of each path score (scores 2 and 3 added together). ( n = 7–8/group). Statistical analysis with Mann-Whitney test. (E) Immunofluorescent staining of cytokeratin-8 (K8) and p63. 52-week-old Cdk12 pc−/− image shows an area of focal HGPIN with expansion of p63(+) BCs. Scale bars in left (100 μm) and right images (50 μm). (F) Ki67 immunohistochemistry in Cdk12 pc−/− or WT mice. Scale bars, 50 μm. Bar graph indicates percentage Ki67(+) cells per high-powered field ( n = 9 images from 3 mice/group). Data represented as mean ± SEM. (G) Immunohistochemistry for immune cell markers, indicating T cell-predominant infiltrate surrounding lesions in Cdk12 pc−/− animals. Scale bars, 100 μm. Bar graph data indicate number of each cell type per high-powered field ( n = 3–5 images from 3 mice/group). Box indicates standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Student’s t test used in (C), (F), and (G). See also Figure S1 .

Article Snippet: Rabbit polyclonal anti-CDK12 , Sigma-Aldrich , Cat# HPA008038; RRID: AB_1078570.

Techniques: Staining, Immunohistochemistry, MANN-WHITNEY, Standard Deviation

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: Organoids derived from the Cdk12 pc−/− prostate are morphologically abnormal, with impaired basal-luminal segregation (A and B) Images of organoids derived from Pb-Cre ; Cdk12 f/f ; mT/mG prostate BCs (52-week time point). Tom indicates Td-tomato-expressing cells with wild-type Cdk12 ( Cdk12 WT ). GFP indicates GFP-expressing cells with Cdk12 ablation ( Cdk12 KO ). Scale bars, 500 μm in (A) and 250 μm in (B). (C) CDK12 immunoblot in Cdk12 WT vs. Cdk12 KO organoids. (Vinculin, loading control). (D) Cdk12 KO organoid morphology: H&E staining, and CDK12 immunohistochemistry. Scale bars 200 μm in top and 50 μm in bottom panels. (E) Immunofluorescence for cytokeratin-8 (K8) and p63 indicating basal-luminal disorganization in Cdk12 KO organoids. Scale bars, 200 μm. (F) Uniform Manifold Approximation and Projection (UMAP) of scRNA-seq from Cdk12 WT organoids ( n = 3). The five identified cell states progress from Basal_1, Basal_2, Basal_3, Lum_1, to Lum_2. (G) UMAP of scRNA-seq from Cdk12 KO organoids ( n = 3). (H) Cells from Cdk12 KO organoids ( n = 3) projected into the UMAP of Cdk12 WT . Pseudocolor indicates presence (yellow) or absence (purple) of Cdk12 transcript. (I) Distributions of different cell states in Cdk12 WT and Cdk12 KO organoids. The most differentiated (Lum_2) population is lost in Cdk12 KO organoids. (J) GSEA for human CDK12 -loss signature—shared down-regulated genes from human PCa with CDK12 inactivation and siCDK12 knockdown LNCaP cells (18)— in Cdk12 KO organoids. Heatmap of logFC ( Cdk12 KO vs. Cdk12 WT organoids) for genes in signature. Genes contributing to negative enrichment (leading edge) are labeled. See also Figure S2 .

Article Snippet: Rabbit polyclonal anti-CDK12 , Sigma-Aldrich , Cat# HPA008038; RRID: AB_1078570.

Techniques: Derivative Assay, Expressing, Western Blot, Control, Staining, Immunohistochemistry, Immunofluorescence, Knockdown, Labeling

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: Cdk12 and Trp53 inactivating alterations interact to promote PCa (A) Workflow for CRISPR library screening of Cdk12 -interacting genes. (B) Snake plot representing log 2 fold change of guide RNAs in sequenced tumor samples described in (A). ( n = 3/group in 2 unique experiments). (C) Immunohistochemistry for p53 (left panels) and γH2AX (right panels) in prostates of one-year-old WT and Cdk12 pc−/− mice. Scale bars, 50 μm. Bar graph indicates percent γH2AX(+) cells from average of 3–5 sections from each of 3 mice. Data represented as mean ± SEM. t test used for individual comparisons. (D) Protein expression of p53 and γH2AX in Cdk12 WT and Cdk12 KO organoids (GAPDH, loading control). (E) CDK12-p53 co-staining in Cdk12 WT and Cdk12 KO organoids. Scale bars, 50 μm. (F) CRISPR-mediated Trp53 ablation in Cdk12 WT and Cdk12 KO organoids. sgp53 indicates Trp53 -specific guide RNA. sgNT indicates control non-targeting guide RNA. (α-tubulin, loading control). (G) Relative expression (Rel Exp) levels of Trp53 and p53 target genes in samples described in (F). ( n = 3/group). Data represented as mean ± SEM. One-way AVOVA used for statistical comparisons. (H) Cell proliferation in organoids from groups indicated in (F) measured by CellTiter-Glo (CTG assay). ( n = 3–4/group). Data represented as mean ± SEM. Two-way ANOVA used for statistical comparisons. (I) Kaplan-Meier plots indicating tumor formation during 70 days post-implantation of Cdk12 WT and Cdk12 KO organoids with or without Trp53 ablation ( n = 10/group). (J) Immunohistochemistry of AR, p53, CDK12, and γH2AX in Cdk12 KO -sgp53 allografts. Scale bars, 50 μm. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also Figure S3 and Table S1 .

Article Snippet: Rabbit polyclonal anti-CDK12 , Sigma-Aldrich , Cat# HPA008038; RRID: AB_1078570.

Techniques: CRISPR, Library Screening, Immunohistochemistry, Expressing, Control, Staining, CTG Assay

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: Cdk12/Trp53 double knockout allografts exhibit lymphocytic immune responses and increased sensitivity to ICB therapy (A) Growth of Cdk12 KO -sgp53, Myc-CaP, and TRAMP-C2 allografts in immunocompetent wild-type mice. ( n = 10–15/group). (B) Immunohistochemistry of CDK12, T cell markers (CD3, CD4, CD8), and natural killer cell marker granzyme B in Cdk12 KO -sgp53 allografts, Myc-CaP allografts, and TRAMP-C2 allografts, and prostates of Pten pc−/− PCa mouse model. Scale bars, 50 μm. (C and D) Tumor growth curve and endpoint weights of Cdk12 KO -sgp53 (C) and TRAMP-C2 (D) allografts treated with anti-PD1/CTLA4 cocktail. ( n = 8–14/group). (E) Flow cytometry-based quantification of CD4(+) and CD8(+) T cells (total, IFNγ(+), granzyme B(+)) in Cdk12 KO -sgp53 and TRAMP-C2 allograft samples +/− treatment with anti-PD1/CTLA4 cocktail. ( n = 7–8/group). (F) Kaplan-Meier plots demonstrating survival of prostate-specific Pten pc−/− and Pten pc−/− Cdk12 pc−/− mice. (G) Genitourinary tract weights of Pten pc−/− and Pten pc−/− Cdk12 pc−/− mice as well as wild-type mice (52 weeks). (H) Cell proliferation in complete media of epithelial cell organoids derived from Pten pc−/− and Pten pc−/− Cdk12 pc−/− mice measured by CTG assay. ( n = 4/group). Data represented as mean ± SEM. Data represented as mean ± SEM. One-way ANOVA for multiple comparisons (G), two-way ANOVA for multiple variables (C) and (E), and unpaired t test was used for tumor weight in (C) and (D). ∗∗∗∗ p < 0.0001; ns, not significant. See also Figures S4 and .

Article Snippet: Rabbit polyclonal anti-CDK12 , Sigma-Aldrich , Cat# HPA008038; RRID: AB_1078570.

Techniques: Double Knockout, Immunohistochemistry, Marker, Flow Cytometry, Derivative Assay, CTG Assay

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: Cdk12 ablation increases AR- and MYC-mediated signaling and promotes TRCs (A) Protein expression of CDK12, AR, and FOXA1 in multiple monoclonal Cdk12 WT and Cdk12 KO organoid lines. (GAPDH, loading control). (B) Gene set enrichment of AR target genes (activated and repressed) in Cdk12 KO organoids compared to Cdk12 WT . (C) Proliferation of Cdk12 WT and Cdk12 KO organoids grown in the absence of epidermal growth factor (EGF) and dihydrotestosterone (DHT) as measured by the CTG assay. ( n = 3 replicates per group in 2 unique experiments). (D and E) Morphology and viability quantification of Cdk12 WT and Cdk12 KO organoids subjected to enzalutamide (Enza) treatment. ( n = 3 replicates per group in 2 unique experiments). ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant. (F) Protein expression of CDK12, MYC, BRD4, BRD3, and BRD2 in Cdk12 WT and Cdk12 KO organoid lines. (G) Gene set enrichment of MYC target genes in Cdk12 KO organoids compared to Cdk12 WT . (H) Morphology of Cdk12 WT and Cdk12 KO organoid lines treated with JQ1 (1 μM). ( n = 3/group in 2 unique experiments). (I) Viability curves and IC 50 values for JQ1-treated Cdk12 WT and Cdk12 KO organoid lines. (J) Dot blot analysis quantifying R-loops in Cdk12 WT and Cdk12 KO organoids. RNase H1 treatment serves as a negative control. (K) Immunofluorescence images of R-loop (red) staining of Cdk12 WT and Cdk12 KO organoids (left) and quantification of fluorescence intensity (right). 100–200 cells/group. (L) Experimental workflow for identification of TRCs. Briefly, 2.5 mM of Thymidine was used to synchronize the cells, and 75 μM of DRB was used to inhibit transcription. (M) Representative immunofluorescence images of γH2AX staining in organoids treated as described in (L). (N) Quantification of γH2AX-positive cells in (M); ( n = 6/group, 3 unique experiments conducted). (O) Representative immunofluorescence images of γH2AX staining in unsynchronized organoids. (P) Quantification of γH2AX-positive cells in (O); n = 6–8 per group (3 unique experiments conducted). (Q) Detection of TRC by PLA assay. (R) Quantification of PLA foci per nucleus in (Q); 100–400 cells analyzed per group (2 unique experiments conducted). Data represented as mean ± SEM. One-way ANOVA for multiple comparisons, two-way ANOVA for multiple variables.

Article Snippet: Rabbit polyclonal anti-CDK12 , Sigma-Aldrich , Cat# HPA008038; RRID: AB_1078570.

Techniques: Expressing, Control, CTG Assay, Dot Blot, Negative Control, Immunofluorescence, Staining, Fluorescence

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: Cdk12 KO organoids and CDK12 -mutant tumors are preferentially sensitive to a CDK13/12 degrader (A) Snake plot representing data from siRNA screen for CDK12 synthetic lethal effects via 1NM sensitivity in CDK12 as cells. Negative Z scores indicate CDK12 synthetic lethal effects, with CDK13 representing most profound effect. (B) Immunoblot indicating CDK13 gene silencing with two different siRNAs (siCDK13.1 and siCDK13.2). (C) Curve depicting cell survival in 1NM-exposed CDK12 as cells transfected with one of two unique CDK13 siRNAs (siCDK13.1 and siCDK13.2) or control siRNA (siCON). (D) CRISPR-mediated Cdk13 (sgCdk13(1 + 3), or sgCdk13(2 + 4)) knockout in Cdk12 WT and Cdk12 KO organoids harvested on day 5 after lentiviral transduction. Protein expression of CDK12 and CDK13 in organoids (Vinculin, loading control). (E) Bright-field images of organoids described in (D). Scale bars, 200 μm. (F) Relative growth quantification from images in (E). ( n = 3/group). (G) CRISPR ablation of Cdk12 (sgCdk12) and Cdk13 (sgCdk13(1 + 3), or sgCdk13(2 + 4)) in Myc-CaP cells. Protein expression of CDK12 and CDK13 in Myc-CaP cells treated with indicated sgRNAs. (H) Colony formation assay showing survival in cells treated with indicated sgRNAs (representative data from 3 unique experiments). (I) Relative growth quantification from images in (H) (analysis of 11 high-powered fields per sample over 2 unique experiments). (J) (Top panel) C4-2B cells subjected to CRISPR-based CDK12 ablation ( CDK12 KO) or control sgRNA (C4-2B CTRL): percent confluence with siRNA-based CDK13 knockdown (si CDK13 ) or control siRNA (siNTC). (Bottom panel) C4-2B CDK12 KO and C4-2B CTRL cells: percent confluence with siRNA-based CCNK knockdown or control siRNA treatment. ( n = 3/group). (K) Images of Cdk12 WT and Cdk12 KO organoids (with or without Trp53 ablation) following treatment with CDK12/13 degrader (YJ9069). sgp53 indicates Trp53 ablation, while sgNT indicates intact Trp53 . Scale bars, 1,000 μm. (L) Viability curves and IC 50 values for YJ9069 treatment of groups described in (K). ( n = 4) (M) Protein expression of p-Ser RNA Pol-II, CDK12, CDK13, and p53 in Cdk12 WT and Cdk12 KO organoids with or without Trp53 ablation subjected to YJ9069 degrader or vehicle treatment. (N) IC 50 of organoids derived from PDX lines with WT CDK12 (MDA153, MDA146-12, LuCaP23.1, LuCaP86.2, LuCaP96, PC295) and inactivating CDK12 mutation (LTL706B, MDA117, MDA328). ( n = 3 per line). Data represented as mean ± SEM. One-way ANOVA for multiple comparisons, two-way ANOVA for multiple variables, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. See also Figures S6 , and Table S2 .

Article Snippet: Rabbit polyclonal anti-CDK12 , Sigma-Aldrich , Cat# HPA008038; RRID: AB_1078570.

Techniques: Mutagenesis, Western Blot, Transfection, Control, CRISPR, Knock-Out, Transduction, Expressing, Colony Assay, Knockdown, Derivative Assay

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet: CDK13/12 degrader inhibits CDK12 -mutant tumor growth in vivo (A) In vivo treatment of Cdk12 KO -sgp53 allografts with YJ9069 or vehicle: line graph indicates tumor volume normalized to baseline. Bar graph indicates tumor weight at endpoint. ( n = 9–10 mice, each with 2 tumors, per group) (B) In vivo treatment of TRAMP-C2 allografts with YJ9069 or vehicle: graphs as indicated in (A) ( n = 9–10 mice, each with 2 tumors, per group). (C and D) Unmodified (sgNT-treated) Myc-CaP allografts (C) or sgCdk12-treated Myc-CaP allografts (D) subjected to in vivo YJ9069 treatment: line graphs indicate tumor volume. Bar graphs indicate tumor weight at end of treatment time course. ( n = 6–9/group). (E–H) CDK12 immunohistochemistry and TUNEL staining of unmodified (sgNT-treated) Myc-CaP allografts (E) and sgCdk12-treated Myc-CaP allografts (F). Bar graphs (G and H) indicate quantification of TUNEL(+) cells per high-powered field (scale bars, 50 μm). (I and J) YJ9069 treatment of subcutaneously implanted PDX lines, LTL706B ( CDK12 -mutant), and MDA146-12 (intact CDK12 ). Graphs indicate tumor volume ( n = 8–9 mice, each with 2 tumors per group). Two-way ANOVA used for tumor volume in (A), (D), and (I); unpaired t test used for tumor weight in (A–D) and (I and J) and TUNEL staining (G and H). ∗∗∗∗ p < 0.0001; ns, not significant. See also Figure S7 .

Article Snippet: Rabbit polyclonal anti-CDK12 , Sigma-Aldrich , Cat# HPA008038; RRID: AB_1078570.

Techniques: Mutagenesis, In Vivo, Immunohistochemistry, TUNEL Assay, Staining

Journal: Cell Reports Medicine

Article Title: CDK12 loss drives prostate cancer progression, transcription-replication conflicts, and synthetic lethality with paralog CDK13

doi: 10.1016/j.xcrm.2024.101758

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CDK12 , Sigma-Aldrich , Cat# HPA008038; RRID: AB_1078570.

Techniques: Virus, Recombinant, Plasmid Preparation, Blocking Assay, Lysis, Protease Inhibitor, Membrane, Transfection, SYBR Green Assay, Cell Viability Assay, Avidin-Biotin Assay, Software

Journal: bioRxiv

Article Title: CDK12/CDK13 inhibition disrupts a transcriptional program critical for glioblastoma survival

doi: 10.1101/2023.07.14.548985

Figure Lengend Snippet: ( A ) Dose-response curves from MTT assays eleven high-grade glioma and seven non-glioma cell lines treated with THZ531. Data represent mean ± SD of three replicates. ( B ) Bar graph showing IC50 values from the MTT assays in (A). ( C ) in vitro cell proliferation assay of the GSCs G7 and G144, and HeLa cells treated as indicated. Data represent mean ± SD of three replicates. ( D ) Clonogenic survival assay of G7, G144 and HeLa cells treated as indicated. ( E ) Competition assays of sgRNAs targeting CDK9, CDK12 and CDK13 as well as positive controls (essential genes, MCM2 and RPS19). Non-targeting sgRNA (NC) was used as negative control. ( F ) Boxplot representing average migration speed of four high-grade glioma cells treated as indicated. The EGFR inhibitor Gefitinib was used as positive control. Each data-point boxplots represents average migration speeds in 4-6 acquired time-lapse movies. The boxplot is representative of three independent experiments. *p < 0.01, **p < 0.001.

Article Snippet: The staining procedure included heat and chemical treatment of the slides with EnVision FLEX TRS at low pH at 97°C (20 min), incubation (30 min) with polyclonal rabbit-anti-human-CDK12 antibody (ab246887; Abcam; 1:50) and 3 min endogenous enzyme block with EnV FLEX Peroxidase-Blocking solution.

Techniques: In Vitro, Proliferation Assay, Clonogenic Cell Survival Assay, Negative Control, Migration, Positive Control

Journal: bioRxiv

Article Title: CDK12/CDK13 inhibition disrupts a transcriptional program critical for glioblastoma survival

doi: 10.1101/2023.07.14.548985

Figure Lengend Snippet: ( A ) Representative images of CDK12 immunohistochemistry in control and glioblastoma tissue. The top row shows control stainings whereas the bottom row left to right reflect CDK12 expression in three different glioblastoma patients (for information on patients, see ). Scale bar 50 μM. ( B ) Representative image showing effect of different inhibitors with indicated doses on glioblastoma organoids. ( C ) Dose-response curve showing data from glioblastoma organoids treated with inhibitors as indicated and measured for cell viability 72 h post treatment. ( D ) Dot-plot showing IC50 values from the assays in ( C ).

Article Snippet: The staining procedure included heat and chemical treatment of the slides with EnVision FLEX TRS at low pH at 97°C (20 min), incubation (30 min) with polyclonal rabbit-anti-human-CDK12 antibody (ab246887; Abcam; 1:50) and 3 min endogenous enzyme block with EnV FLEX Peroxidase-Blocking solution.

Techniques: Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: CDK12/CDK13 inhibition disrupts a transcriptional program critical for glioblastoma survival

doi: 10.1101/2023.07.14.548985

Figure Lengend Snippet: (A) Representative images of CDK12 immunohistochemistry in cortex/infiltration zone/cell-rich tumor of glioblastoma patients. Nuclear CDK12 expression absent in cortex areas without obvious tumor cell infiltration (top) while the number of CDK12-positive cells increases with tumor cell density in the infiltration zone (CDK12-positive cells = black arrowhead; CDK12-negative cortical neurons = blue arrowhead). Multinucleated giant cells (asterisk, bottom) in highly cellular areas expressing CDK12. Scale bar 50 µm. (B) Summary of the patient characteristics of the ex vivo GBM organoids. (C) Four high-grade glioma cell lines were treated with increasing doses of inhibitors as indicated. After 72h, the cell viability was assessed using Cell-Titer-Glo. Graph displays a dose-response curve with percent cell viability relative to the DMSO control for each cell line. Data represent mean ± SD of three replicates. (D) Dot-plot showing IC50 values for dose response of inhibitors on a panel of GSCs shown in (C).

Article Snippet: The staining procedure included heat and chemical treatment of the slides with EnVision FLEX TRS at low pH at 97°C (20 min), incubation (30 min) with polyclonal rabbit-anti-human-CDK12 antibody (ab246887; Abcam; 1:50) and 3 min endogenous enzyme block with EnV FLEX Peroxidase-Blocking solution.

Techniques: Immunohistochemistry, Expressing, Ex Vivo

Journal: Cell reports

Article Title: HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity

doi: 10.1016/j.celrep.2021.109514

Figure Lengend Snippet: (A) FLAG-tagged SERINC5 protein was expressed in HEK293T cells and purified by anti-FLAG M2 affinity chromatography. After SDS-PAGE, proteins from total cell lysate and three eluted fractions were analyzed after being stained with Coomassie brilliant blue. The monomeric SERINC5 band is labeled. (B) FLAG-tagged SERINC5 was expressed with a WT or ΔNef HIV-1 proviral vector in HEK293T cells and purified and analyzed similar to as in (A). The monomeric SERINC5 band is labeled and a protein band at ~170 kDa is indicated by an asterisk. (C) purified proteins from (B) were analyzed by liquid chromatography-mass spectrometry (LC-MS). Control experiments were conducted using beads that were not conjugated with any antibodies. Six proteins with molecular masses of 150–200 kDa that are not found in the control experiments are listed. (D) FLAG-tagged CycK was expressed with WT or ΔNef HIV-1 in HEK293T cells. Proteins were immunoprecipitated with an anti-FLAG antibody and analyzed by western blotting (WB). CycK was detected by an anti-FLAG antibody and Nef and GAPDH were detected by their specific antibodies. IP, immunoprecipitation; Input, cell lysate. (E) FLAG-tagged CycK was expressed with HA-tagged CDK12 or CDK13 in HEK293T cells. Proteins were immunoprecipitated and analyzed as in (D). CDK12 and CDK13 were detected by an anti-HA antibody. (F) FLAG-tagged CDK13 was expressed with HA-tagged SERINC5 in the presence of WT or ΔNef HIV-1 in HEK293T cells. Proteins were immunoprecipitated and analyzed as in (D) and (E). All experiments were repeated twice, and similar results were obtained.

Article Snippet: Horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-FLAG, anti-HA antibodies, and anti-actin were purchased from Sigma; HRP-conjugated mouse monoclonal anti-GAPDH was purchased from Proteintech; rabbit polyclonal anti-CrkRS (CDK12) and anti-CDC2L5 (CDK13) were purchased from Novus; rabbit polyclonal anti-CycK was purchased from Abcam; HRP-conjugated anti-human, -rabbit, and -mouse immunoglobulin G secondary antibodies were purchased from Thermo Fisher.

Techniques: Purification, Affinity Chromatography, SDS Page, Staining, Labeling, Plasmid Preparation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Western Blot

Journal: Cell reports

Article Title: HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity

doi: 10.1016/j.celrep.2021.109514

Figure Lengend Snippet: (A) SERINC5 was expressed with Cas9 and CCNK -gRNA expression vectors in the presence of WT or ΔNef HIV-1 in HEK293T (upper gels) or SERINC3/5 knockout Jurkat-TAg cells (lower gels) and analyzed by WB. SERINC5 was detected by an anti-FLAG antibody. (B) SERINC5 with an internal HA tag (Ser5-iHA) was expressed with a CCNK -shRNA expression vector in the presence of WT or ΔNef HIV-1 in HEK293T cells. SERINC5 expression on the cell surface was analyzed by flow cytometry using a fluorescent anti-HA antibody. Results are presented as histograms and levels of SERINC5-positive cells are indicated (%). (C) SERINC5 was expressed with a CDK12 - or CDK13 -shRNA expression vector in the presence of WT or ΔNef HIV-1 in HEK293T cells and analyzed by WB. SERINC5 was detected by an anti-FLAG antibody. (D) Ser5-iHA was expressed with a CDK12 - or CDK13 -shRNA expression vector as in (B) and analyzed similarly. (E) Mean fluorescence intensity (MFI) values for the SERINC5-positive cell populations in (B) and (D) were statistically analyzed. (F) SERINC5 was expressed with WT or ΔNef HIV-1 in the presence of an indicated shRNA or control (Ctrl) expression vector in HEK293T cells. After virions were collected and quantified by p24 Gag ELISA, TZM-bI cells were infected with an equal amount of virions and viral infectivity was analyzed by measuring the intracellular firefly luciferase activity after 48 h of infection. Infectivity is presented as a relative value, with the WT HIV-1 infectivity in the absence of SERINC5 set to 100%. (G) WT and ΔNef HIV-1 were produced from HEK293T cells in the presence of a CCNK -, CDK13 -, or CDK12 -shRNA expression vector and viral infectivity was analyzed and presented as in (F). Error bars in (E)–(G) represent SEM from three independent experiments. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant (p > 0.05).

Article Snippet: Horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-FLAG, anti-HA antibodies, and anti-actin were purchased from Sigma; HRP-conjugated mouse monoclonal anti-GAPDH was purchased from Proteintech; rabbit polyclonal anti-CrkRS (CDK12) and anti-CDC2L5 (CDK13) were purchased from Novus; rabbit polyclonal anti-CycK was purchased from Abcam; HRP-conjugated anti-human, -rabbit, and -mouse immunoglobulin G secondary antibodies were purchased from Thermo Fisher.

Techniques: Expressing, Knock-Out, shRNA, Plasmid Preparation, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Infection, Luciferase, Activity Assay, Produced

Journal: Cell reports

Article Title: HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity

doi: 10.1016/j.celrep.2021.109514

Figure Lengend Snippet: (A) SERINC5 was expressed with the indicated CDK13 or CDK12 proteins in the presence of WT or ΔNef HIV-1 in HEK293T cells. SERINC5, CDK13, and CDK12 proteins were detected by WB using an anti-FLAG antibody. (B) SERINC5 was expressed with WT or ΔNef HIV-1 in HEK293T cells and treated with THZ531 at 100 μM. SERINC5 expression was analyzed by WB using an anti-FLAG antibody. (C) SERINC5 and SERINC5 S360A were expressed with WT or ΔNef HIV-1 in HEK293T cells and purified similar to as previously. After SDS-PAGE, proteins were analyzed after being stained with Coomassie brilliant blue or Phos-tag. (D) GST-ICL4 and GST-ICL4 S360A recombinant proteins were expressed in E. coli and purified by glutathione resin. An in vitro kinase assay was conducted by incubating these proteins with affinity-purified CycK/CDK13. After SDS-PAGE, proteins were analyzed after being stained with Coomassie brilliant blue or Phos-tag. (E) Peptide 357 FCFSPGGEDTEEQQPGK 373 , its S360-phosphorylated version, and its T366K-substituted version were synthesized. They were analyzed by high-resolution MS via direct infusion or analyzed by CZE-MS after treatment with affinity-purified CycK/CDK13.

Article Snippet: Horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-FLAG, anti-HA antibodies, and anti-actin were purchased from Sigma; HRP-conjugated mouse monoclonal anti-GAPDH was purchased from Proteintech; rabbit polyclonal anti-CrkRS (CDK12) and anti-CDC2L5 (CDK13) were purchased from Novus; rabbit polyclonal anti-CycK was purchased from Abcam; HRP-conjugated anti-human, -rabbit, and -mouse immunoglobulin G secondary antibodies were purchased from Thermo Fisher.

Techniques: Expressing, Purification, SDS Page, Staining, Recombinant, In Vitro, Kinase Assay, Affinity Purification, Synthesized

Journal: Cell reports

Article Title: HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity

doi: 10.1016/j.celrep.2021.109514

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-FLAG, anti-HA antibodies, and anti-actin were purchased from Sigma; HRP-conjugated mouse monoclonal anti-GAPDH was purchased from Proteintech; rabbit polyclonal anti-CrkRS (CDK12) and anti-CDC2L5 (CDK13) were purchased from Novus; rabbit polyclonal anti-CycK was purchased from Abcam; HRP-conjugated anti-human, -rabbit, and -mouse immunoglobulin G secondary antibodies were purchased from Thermo Fisher.

Techniques: Recombinant, Protease Inhibitor, Mutagenesis, Clone Assay, Luciferase, Staining, Knock-Out, Marker, Software

Journal: Cell reports

Article Title: HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity

doi: 10.1016/j.celrep.2021.109514

Figure Lengend Snippet: (A) FLAG-tagged SERINC5 protein was expressed in HEK293T cells and purified by anti-FLAG M2 affinity chromatography. After SDS-PAGE, proteins from total cell lysate and three eluted fractions were analyzed after being stained with Coomassie brilliant blue. The monomeric SERINC5 band is labeled. (B) FLAG-tagged SERINC5 was expressed with a WT or ΔNef HIV-1 proviral vector in HEK293T cells and purified and analyzed similar to as in (A). The monomeric SERINC5 band is labeled and a protein band at ~170 kDa is indicated by an asterisk. (C) purified proteins from (B) were analyzed by liquid chromatography-mass spectrometry (LC-MS). Control experiments were conducted using beads that were not conjugated with any antibodies. Six proteins with molecular masses of 150–200 kDa that are not found in the control experiments are listed. (D) FLAG-tagged CycK was expressed with WT or ΔNef HIV-1 in HEK293T cells. Proteins were immunoprecipitated with an anti-FLAG antibody and analyzed by western blotting (WB). CycK was detected by an anti-FLAG antibody and Nef and GAPDH were detected by their specific antibodies. IP, immunoprecipitation; Input, cell lysate. (E) FLAG-tagged CycK was expressed with HA-tagged CDK12 or CDK13 in HEK293T cells. Proteins were immunoprecipitated and analyzed as in (D). CDK12 and CDK13 were detected by an anti-HA antibody. (F) FLAG-tagged CDK13 was expressed with HA-tagged SERINC5 in the presence of WT or ΔNef HIV-1 in HEK293T cells. Proteins were immunoprecipitated and analyzed as in (D) and (E). All experiments were repeated twice, and similar results were obtained.

Article Snippet: Horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-FLAG, anti-HA antibodies, and anti-actin were purchased from Sigma; HRP-conjugated mouse monoclonal anti-GAPDH was purchased from Proteintech; rabbit polyclonal anti-CrkRS (CDK12) and anti-CDC2L5 (CDK13) were purchased from Novus; rabbit polyclonal anti-CycK was purchased from Abcam; HRP-conjugated anti-human, -rabbit, and -mouse immunoglobulin G secondary antibodies were purchased from Thermo Fisher.

Techniques: Purification, Affinity Chromatography, SDS Page, Staining, Labeling, Plasmid Preparation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Western Blot

Journal: Cell reports

Article Title: HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity

doi: 10.1016/j.celrep.2021.109514

Figure Lengend Snippet: (A) SERINC5 was expressed with Cas9 and CCNK -gRNA expression vectors in the presence of WT or ΔNef HIV-1 in HEK293T (upper gels) or SERINC3/5 knockout Jurkat-TAg cells (lower gels) and analyzed by WB. SERINC5 was detected by an anti-FLAG antibody. (B) SERINC5 with an internal HA tag (Ser5-iHA) was expressed with a CCNK -shRNA expression vector in the presence of WT or ΔNef HIV-1 in HEK293T cells. SERINC5 expression on the cell surface was analyzed by flow cytometry using a fluorescent anti-HA antibody. Results are presented as histograms and levels of SERINC5-positive cells are indicated (%). (C) SERINC5 was expressed with a CDK12 - or CDK13 -shRNA expression vector in the presence of WT or ΔNef HIV-1 in HEK293T cells and analyzed by WB. SERINC5 was detected by an anti-FLAG antibody. (D) Ser5-iHA was expressed with a CDK12 - or CDK13 -shRNA expression vector as in (B) and analyzed similarly. (E) Mean fluorescence intensity (MFI) values for the SERINC5-positive cell populations in (B) and (D) were statistically analyzed. (F) SERINC5 was expressed with WT or ΔNef HIV-1 in the presence of an indicated shRNA or control (Ctrl) expression vector in HEK293T cells. After virions were collected and quantified by p24 Gag ELISA, TZM-bI cells were infected with an equal amount of virions and viral infectivity was analyzed by measuring the intracellular firefly luciferase activity after 48 h of infection. Infectivity is presented as a relative value, with the WT HIV-1 infectivity in the absence of SERINC5 set to 100%. (G) WT and ΔNef HIV-1 were produced from HEK293T cells in the presence of a CCNK -, CDK13 -, or CDK12 -shRNA expression vector and viral infectivity was analyzed and presented as in (F). Error bars in (E)–(G) represent SEM from three independent experiments. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant (p > 0.05).

Article Snippet: Horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-FLAG, anti-HA antibodies, and anti-actin were purchased from Sigma; HRP-conjugated mouse monoclonal anti-GAPDH was purchased from Proteintech; rabbit polyclonal anti-CrkRS (CDK12) and anti-CDC2L5 (CDK13) were purchased from Novus; rabbit polyclonal anti-CycK was purchased from Abcam; HRP-conjugated anti-human, -rabbit, and -mouse immunoglobulin G secondary antibodies were purchased from Thermo Fisher.

Techniques: Expressing, Knock-Out, shRNA, Plasmid Preparation, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Infection, Luciferase, Activity Assay, Produced

Journal: Cell reports

Article Title: HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity

doi: 10.1016/j.celrep.2021.109514

Figure Lengend Snippet: (A) SERINC5 was expressed with the indicated CDK13 or CDK12 proteins in the presence of WT or ΔNef HIV-1 in HEK293T cells. SERINC5, CDK13, and CDK12 proteins were detected by WB using an anti-FLAG antibody. (B) SERINC5 was expressed with WT or ΔNef HIV-1 in HEK293T cells and treated with THZ531 at 100 μM. SERINC5 expression was analyzed by WB using an anti-FLAG antibody. (C) SERINC5 and SERINC5 S360A were expressed with WT or ΔNef HIV-1 in HEK293T cells and purified similar to as previously. After SDS-PAGE, proteins were analyzed after being stained with Coomassie brilliant blue or Phos-tag. (D) GST-ICL4 and GST-ICL4 S360A recombinant proteins were expressed in E. coli and purified by glutathione resin. An in vitro kinase assay was conducted by incubating these proteins with affinity-purified CycK/CDK13. After SDS-PAGE, proteins were analyzed after being stained with Coomassie brilliant blue or Phos-tag. (E) Peptide 357 FCFSPGGEDTEEQQPGK 373 , its S360-phosphorylated version, and its T366K-substituted version were synthesized. They were analyzed by high-resolution MS via direct infusion or analyzed by CZE-MS after treatment with affinity-purified CycK/CDK13.

Article Snippet: Horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-FLAG, anti-HA antibodies, and anti-actin were purchased from Sigma; HRP-conjugated mouse monoclonal anti-GAPDH was purchased from Proteintech; rabbit polyclonal anti-CrkRS (CDK12) and anti-CDC2L5 (CDK13) were purchased from Novus; rabbit polyclonal anti-CycK was purchased from Abcam; HRP-conjugated anti-human, -rabbit, and -mouse immunoglobulin G secondary antibodies were purchased from Thermo Fisher.

Techniques: Expressing, Purification, SDS Page, Staining, Recombinant, In Vitro, Kinase Assay, Affinity Purification, Synthesized

Journal: Cell reports

Article Title: HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity

doi: 10.1016/j.celrep.2021.109514

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-FLAG, anti-HA antibodies, and anti-actin were purchased from Sigma; HRP-conjugated mouse monoclonal anti-GAPDH was purchased from Proteintech; rabbit polyclonal anti-CrkRS (CDK12) and anti-CDC2L5 (CDK13) were purchased from Novus; rabbit polyclonal anti-CycK was purchased from Abcam; HRP-conjugated anti-human, -rabbit, and -mouse immunoglobulin G secondary antibodies were purchased from Thermo Fisher.

Techniques: Recombinant, Protease Inhibitor, Mutagenesis, Clone Assay, Luciferase, Staining, Knock-Out, Marker, Software