Journal: Journal of Biological Chemistry

Article Title: Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition

doi: 10.1074/jbc.m109.096552

Figure Lengend Snippet: FIGURE 3. Partially dephosphorylated Tyr(P)15 Cdk1 is catalytically active. A, Cdk1 was immunoprecipitated from 500 g of lysates of U2OS Cdc25A cells, collectedat10hafterrelease.Samplesweresplitandusedforimmunoblotting(right)orkinaseassays(left).NotethehighlevelsofTyr15(pTyr15)phosphorylationand Cdk1kinaseactivityininducedcells.B,samelysatesasinAwereusedforIPwiththeanti-Tyr(P)15 oranti-Cdk1antibodyandsplitforsubsequentH1kinaseassaysand WB (G). C, quantifications of relative kinase activities of Cdk1 in non-induced and induced U2OS cells. The kinase was immunoprecipitated using anti-Cdk1 or anti-Tyr(P)15 antibodies. Activity in non-induced cells was considered as 1; n 3. Error bars, S.D. D, lysates and Cdk1 immunoprecipitates prepared as in A were immunoblotted with the anti-Tyr(P)15 antibody. E, the same lysates as above were depleted of Cdk2 kinase by two rounds of immunoprecipitations, and efficiency of depletionwascontrolledbyimmunoblotting(right).Afterdepletion,IPwiththeanti-Tyr(P)15 antibodywasperformed,andthekinaseactivityofimmunoprecipitates wasassessedinanH1kinaseassay(left).F,293Tcellswereco-transfectedwithpCDNA3.1()-FLAG-Cdk1WTortheindicatedmutantsandpCDNA3.1()-HA-cyclinB1; amountsofDNAwereequilibratedusingtheemptyvectorpCDNA3.1().After24hpost-transfection,cellswerelysed,andimmunoprecipitationsusingtheanti-FLAG M2 antibody were performed and used for H1 kinase assays. G, immunoprecipitates obtained using the anti-Tyr(P)15 (B) were probed in WB with the indicated antibodies. Note the substantial increase in amounts of cyclin B co-immunoprecipitated with Tyr(P)15 Cdk1 from induced cells.

Article Snippet: For Western blotting and immunoprecipitations, the following antibodies were used: rabbit polyclonal antibodies against Cdc25A (M-191), Cdc25B (C-20), Cdc25C (C-20), and cyclin A (H-432) and mouse monoclonal anti-Cdc25A (F6), anti-Cdc2 (p34), anti-APC3 (AF3.1), and anti-cyclin A (BF683) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); monoclonal anti-cyclin B1 antibody from Upstate Biotechnology; polyclonal anti-Thr(P)14 Cdc2, anti-Tyr(P)15 cdc2, antiThr(P)161 Cdc2, anti-Wee1, and anti-MEK1/2 antibodies from Cell Signaling; anti-Thr(P)244 APC3 fromRockland Inc.; mouse monoclonal antibodies against -tubulin, anti- -actin, antiFLAG (M2), and anti-Cdk7 (MO1.1) from Sigma; and mouse monoclonal anti-HA antibody (Babco) and anti-poly(ADP-ribose) polymerase (BD Biosciences).

Techniques: Immunoprecipitation, Activity Assay, Transfection

Journal: Journal of Biological Chemistry

Article Title: Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition

doi: 10.1074/jbc.m109.096552

Figure Lengend Snippet: FIGURE 6. Repression of Cdc25A or Cdc25B by shRNA impairs Cdk1-cyclin B complex assembly. A, HeLa cells were transfected with plasmids, encoding shRNA targeting Cdc25A or firefly luciferase as a control and, 24 h after transfection, were synchronized by a double thymidine block. After release, cells were collected at different time intervals and used for FACS to confirm synchronization and trace stages of the cell cycle (not shown) and for preparation of lysates. Left, cyclin B was immunoprecipitated from 500 g of lysates using the antibodiesindicated,andproteinswereresolvedonSDS-PAGEandanalyzedbyWB.DensityofCdk1bandswas measured using ImageJ software, and their relative intensities were calculated as the ratio to the density of the Cdk1 band at 9 h after release (mitosis), considered as 1. Results are indicated below the Cdk1 blots. Right, 50 g of lysates used for IP analyzed by WB to monitor repression of Cdc25A and evenness of loading. B, similar to A, but plasmids expressing shRNA against Cdc25B phosphatase were used. C, similar to A and B, but both Cdc25A and Cdc25B were repressed using corresponding shRNA-expressing constructs. D, data of relative density measurements at time point 9 h were used for statistical analysis in three independent experiments. Error bars, S.D.

Article Snippet: For Western blotting and immunoprecipitations, the following antibodies were used: rabbit polyclonal antibodies against Cdc25A (M-191), Cdc25B (C-20), Cdc25C (C-20), and cyclin A (H-432) and mouse monoclonal anti-Cdc25A (F6), anti-Cdc2 (p34), anti-APC3 (AF3.1), and anti-cyclin A (BF683) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); monoclonal anti-cyclin B1 antibody from Upstate Biotechnology; polyclonal anti-Thr(P)14 Cdc2, anti-Tyr(P)15 cdc2, antiThr(P)161 Cdc2, anti-Wee1, and anti-MEK1/2 antibodies from Cell Signaling; anti-Thr(P)244 APC3 fromRockland Inc.; mouse monoclonal antibodies against -tubulin, anti- -actin, antiFLAG (M2), and anti-Cdk7 (MO1.1) from Sigma; and mouse monoclonal anti-HA antibody (Babco) and anti-poly(ADP-ribose) polymerase (BD Biosciences).

Techniques: shRNA, Transfection, Luciferase, Control, Blocking Assay, Immunoprecipitation, Software, Expressing, Construct

Journal: Journal of Biological Chemistry

Article Title: Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition

doi: 10.1074/jbc.m109.096552

Figure Lengend Snippet: FIGURE 7. Overexpression of Cdc25A correlates with increased electrophoretic mobility of Cdk7. A, lysates of U2OS Cdc25A synchronized cells, collected at 10 h after release from a double thymidine block, were analyzed by immunoblotting with the indicated antibodies. Note a shift of the Thr(P)161 (pThr161) band in induced cells, corresponding to Tyr15/Thr161-phosphorylated Cdk1, in comparison with Thr(P)14/Tyr(P)15/Thr(P)161 phosphorylated in non-induced cells, and a slight increase in Thr161 phosphorylation upon overexpression of Cdc25A. B, induced and non-induced U2OS Cdc25A cells were fixed on coverslips 9hafterreleasefromadoublethymidineblock(left).Cellswerestainedwithanti-cyclinB(green)andanti-Cdk1-Thr(P)161(red)antibodies.DNAwasstainedwith Hoechst (blue). Scale bar, 10 m. On the right, relative signal intensities of anti-Thr(P)161 stainings for 50 cells were determined. Error bars, S.D. in three independent experiments. C, nuclear (left) and detergent-resistant fractions (right) from cells synchronized as in A. Extracts from U2OS Cdc25A cells were resolved using SDS-PAGE and probed with denoted antibodies. Note that in detergent-resistant fractions obtained from induced cells, the faster migrating Cdk7 bands are more abundant than in those from non-induced cells. Phospho-Thr161 Cdk1 (upper band) is also increased in these extracts upon Cdc25A overexpression. The lower bands in Thr(P)161 immunoblots correspond to Thr(P)160 Cdk2, also recognized by the antibody used. D, Cdk7 was immunoprecipi- tated from 500 g of lysates prepared as in A and IP were split for immunoblotting (bottom) and kinase assays using catalytically inactive GST-Cdk1 (GST-Cdk1- KD) as a substrate (top). As a control, mouse IgGs were used.

Article Snippet: For Western blotting and immunoprecipitations, the following antibodies were used: rabbit polyclonal antibodies against Cdc25A (M-191), Cdc25B (C-20), Cdc25C (C-20), and cyclin A (H-432) and mouse monoclonal anti-Cdc25A (F6), anti-Cdc2 (p34), anti-APC3 (AF3.1), and anti-cyclin A (BF683) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); monoclonal anti-cyclin B1 antibody from Upstate Biotechnology; polyclonal anti-Thr(P)14 Cdc2, anti-Tyr(P)15 cdc2, antiThr(P)161 Cdc2, anti-Wee1, and anti-MEK1/2 antibodies from Cell Signaling; anti-Thr(P)244 APC3 fromRockland Inc.; mouse monoclonal antibodies against -tubulin, anti- -actin, antiFLAG (M2), and anti-Cdk7 (MO1.1) from Sigma; and mouse monoclonal anti-HA antibody (Babco) and anti-poly(ADP-ribose) polymerase (BD Biosciences).

Techniques: Over Expression, Blocking Assay, Western Blot, Comparison, Phospho-proteomics, SDS Page, Control

Journal: Cancer research

Article Title: STING promotes homeostasis via regulation of cell proliferation and chromosomal stability

doi: 10.1158/0008-5472.CAN-18-1972

Figure Lengend Snippet: (A) Representative images of chromosome analysis performed on G-banded metaphase cells from primary WT and STINGko MEFs in passage 1 that were either mock-irradiated or treated with 6 Gy IR. (B-C) Bar graph representing the percentage of polyploid cells in BrdU/PI double-labeled p53−/− (B) and p21-depleted (C) HCT116 cell lines. (D) Western analyses of cGAS, STING, and phospho CDC2 Tyr15 expression in lysates from WT and STINGko MEFs harvested 48 hours post-exposure to increasing IR doses. (E) WT and STINGko MEFs at 48 hours post-treatment with increasing IR dose, stained for DNA and mitochondrial/cytoplasmic compartments using Draq5 and MitoTracker Red, respectively; scale bar, 10 µm. (F-G) Kinetic analysis of average nuclear area of MEFs (F) and D54 tumor cells (G) stained with Nuclight Red dye measured over time at baseline and in response to IR. (H) WT and STINGko MEFs treated with WEE1 inhibitor MK1775 in combination with IR (6 Gy) were stained for DNA and mitochondrial/cytoplasmic compartments using Draq5 and MitoTracker Red, respectively at 48 hours post-treatment; scale bar, 10 µm. (I-L) Kinetic analysis of STINGko MEFs (I) as well as shSTING D54 (J), HCT116 (K), and SCC61 (L) proliferation in vitro were measured over time in response to WEE1 inhibitor MK1775 ± IR (6 Gy) using the IncuCyte live cell imaging system. In vitro growth curve data are representative of at least three experiments, each with n = 3 per group. P-values were determined using unpaired Student’s t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

Article Snippet: Western blotting antibodies For confirmation of targeted knockdown experiments as well as transient transfection/reconstitution experiments in both murine and human cell lines, the following primary antibodies were used: anti-STING (clone D2P2F; #13647; Cell Signaling Technology), anti-cGAS (#15102 and #31659; Cell Signaling Technology), anti-p21 (ab109199; Abcam), anti-TBK1 (sc-9910; Santa Cruz Biotechnology), anti-phospho-TBK1 Ser172 (clone D52C2; #5483S; Cell Signaling Technology), anti-IRF-3 (clone FL-425; sc-9082; Santa Cruz Biotechnology), anti-phospho IRF3 Ser396 (70R-35220; Fitzgerald Antibodies), anti-STAT1 p84/p91 (clone C-136; sc-464; Santa Cruz Biotechnology), anti- NF-κB p65 (clone D14E12; #8242; Cell Signaling Technology), anti-phospho-NF-κB p65 Ser536 (clone 93H1; #3033; Cell Signaling Technology), anti-FLAG (M2 clone; Sigma), anti-CDC2 (#77055S; Cell Signaling Technology), anti-phospho-CDC2 Tyr15 (clone 10A11; #4539; Cell Signaling Technology), anti-Rb (#9313S; Cell Signaling Technology) anti-phospho-Rb Ser807/811 (clone D20B12; #8516; Cell Signaling Technology), and anti-Actin-HRP (sc-47778; Santa Cruz Biotechnology).

Techniques: Irradiation, Labeling, Western Blot, Expressing, Staining, In Vitro, Live Cell Imaging

Journal: Stem cell research

Article Title: NOSTRIN: A novel modulator of trophoblast giant cell differentiation.

doi: 10.1016/j.scr.2018.07.023

Figure Lengend Snippet: Fig. 6. NOSTRIN influences actin dynamics and promotes Cdk1 phosphorylation in differentiating trophoblast cells. (A) Western blot showing G and F actin fraction of the cytosol from trophoblast cells on day 2.5 of differentiation following transfection with empty vector, Nostrin cDNA or NostrinΔSH3 mutant cDNA. Experiments were performed in three biolo- gical replicates. (B) A histogram measuring G/F ratio calculated by measuring individual relative densities of G and F fractions using ImageJ software. (C) Immunoblot assay to estimate total cellular β-Actin, Gapdh and rpL7 in whole cell lysate of three tras- fected cell samples. (D) Immuno-precipitation (IP) with anti-N-WASP antibody followed by western blotting (WB) using anti-NOSTRIN and anti-Dynamin antibody in day 2.5 differentiated trophoblast cells following transfection with empty vector, Nostrin cDNA or NostrinΔSH3 mutant cDNA. IgG heavy chain detected in each sample show equal loading. The experiment was repeated thrice to ensure reprodu- cibility. For B, values are represented as mean ± SEM of three independent experiments. (E) Immunoblot assay of p-T14-CDK1, p-Y15-CDK1 and CDK1 using transfected cell samples. RpL7 was used as an endogenous control. (F) Quantification of phospho-proteins (T14 and Y15) relative to basal CDK1 levels.(G) Immuno-precipitation (IP) with anti-CDK1 antibody followed by western blotting (WB) using anti-NOSTRIN in day 2.5 differentiated trophoblast cells following transfection with empty vector, Nostrin cDNA or NostrinΔSH3 mutant cDNA. Statistical analysis was performed by one-way ANOVA (n = 3) followed by Newman Keuls test. *p < .05 and **p < .01.

Article Snippet: Anti-Dynamin (2342) and anti-N-WASP (4848) anti-cdc2 (77055), anti-phospho-cdc2-T14 (2543), anti-phospho-cdc2-Y15 (4539), HRP conjugated goat anti-rabbit (7074) antibodies were purchased from Cell Signaling Technology (USA).

Techniques: Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Software, Immunoprecipitation, Control

Journal: PLoS Genetics

Article Title: Ndd1 Turnover by SCF Grr1 Is Inhibited by the DNA Damage Checkpoint in Saccharomyces cerevisiae

doi: 10.1371/journal.pgen.1005162

Figure Lengend Snippet: In the absence of DNA damage, Ndd1 is phosphorylated by Cdk1, which promotes Ndd1 transcriptional activity but also enables SCF Grr1 to recognize Ndd1 as a substrate. Ndd1 is therefore ubiquitinated and subsequently degraded. In response to DNA damage, Swe1 levels are increased, and Cdk1 is inhibited by phosphorylation which lowers its activity several fold. Ndd1 is no longer recognized by Grr1 and is therefore stabilized. Mec1 and Tel1 are important for maintaining high levels of phospho-Cdk1, and therefore help to stabilize Ndd1, either by promoting Swe1 activity or by inhibiting the phosphatase Mih1 (homolog of Cdc25). While Ndd1 is stabilized, its activity is independently inhibited by Rad53-dependent phosphorylation, leading to the accumulation of inhibited Ndd1. We speculate that maintaining this inhibited pool of Ndd1 may allow the cell to rapidly re-enter the cell cycle once the damage is repaired and the checkpoint is turned off.

Article Snippet: Kellogg), and α-phospho-Cdc2 (Y15) (antibody was raised against human Cdc2 but recognizes S . cerevisiaie Cdc28 inhibitory phosphorylation as well, from Cell Signaling Technology #9111).

Techniques: Activity Assay, Phospho-proteomics

Journal: Cell reports. Medicine

Article Title: Development of an orally bioavailable CDK12/13 degrader and induction of synthetic lethality with AKT pathway inhibition.

doi: 10.1016/j.xcrm.2024.101752

Figure Lengend Snippet: Figure 3. YJ9069 suppresses tumor growth in multiple in vivo models of castration-resistant prostate cancer (A) Dose-response curves and IC50 of cells treated with YJ9069. Data are presented as mean ± standard deviation (n = 3) from one of three independent ex- periments. (B) Immunoblot of CDK12, CDK13, and cleaved PARP for castrated VCaP in vivo xenografted tumors after 5 days treatment with YJ9069 (i.v., 30 mg/kg, 3x/week). Tubulin is the loading control. (C) Representative H&E staining and immunohistochemistry for CDK12, cleaved PARP, and TUNEL from the PD5 study in (B) (scale, 50 mm). (D) Tumor volume (measured twice weekly using calipers) in the castrated VCaP model treated with YJ9069 (i.v., 30 mg/kg, 3x/week) (two-sided t test). Data are mean ± standard error of the mean (SEM) (vehicle: n = 10; YJ9069: n = 11). (E) Tumor weights for vehicle and YJ9069 groups from castrated VCaP study (two-sided t test). Data are presented as mean ± SEM. (F) Waterfall plot depicting the change in tumor volume after 18 days of treatment. Response evaluation criteria in solid tumors (RECIST) was used to stratify tumors: progressive disease (PD), at least a 20% increase in tumor size; stable disease (SD), an increase of <20% to a decrease of <30%; partial response (PR), at least a 30% decrease. The vehicle group has 100% PD; the YJ9069 group has 18% SD and 82% PR. (G) Percent body weight measurement showing the effect of vehicle and YJ9069 in the castrated VCaP model throughout the treatment period. Data are pre- sented as mean ± SEM. (H) Representative H&E staining for vehicle- and YJ9069-treated tumors from castrated VCaP xenograft at the endpoint (scale, 200 mm). The inset scale, 50 mm. (I–M) as in (D–H), except in the WA74 patient-derived xenograft (PDX) model with YJ9069 treatment (i.v., 30 mg/kg, 2x/week or 3x/week) (n = 8 per condition). In the waterfall plot, the YJ9069 2x/week group has 14% PD, 43% SD, and 43% PR; the YJ9069 3x/week group has 100% PR. (N–R) as in (D–H), except in the PC310 PDX model with YJ9069 (i.v., 30 mg/kg, 3x/week). In the waterfall plot, the YJ9069 group has 63% PD, 31% SD, and 6% PR. (S) Genitourinary tract measurement for vehicle and YJ9069 groups at noted doses in CD-1 male mouse (two-sided t test). Data are presented as mean ± SEM (n = 4, biological replicates). (T) Representative photographs with matched H&E staining of the genitourinary region from vehicle and YJ9069 (i.v., 30 mg/kg, 3x/week) groups in CD-1 male mouse (scale, 50 mm). See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies CDK12 Proteintech Cat# 26816-1-AP; RRID:AB_2880645 CDK13 Millipore Cat# ABE1860 RNA pol II CTD phospho Ser2 Cell Signaling Technology Cat# 13499S; RRID: RRID: AB_2798238 c-PARP Cell Signaling Technology Cat# 5625S; RRID:AB_10699459 gH2AX Abcam Cat# ab11174; RRID:AB_297813 CCNK Bethyl Laboratories Cat# A301-939A; RRID:AB_1547934 p-AKT (ser473) Cell Signaling Technology Cat# 4060S; RRID:AB_2315049 p-PRAS40 Cell Signaling Technology Cat# 2997S; RRID:AB_2258110 AKT Cell Signaling Technology Cat# 4691S; RRID:AB_915783 PRAS40 Cell Signaling Technology Cat# 2610S; RRID:AB_916206 p-S6 (Ser235/236) Cell Signaling Technology Cat# 2211S; RRID:AB_331679 GSPT1 Proteintech Cat# 10763-1-AP; RRID:AB_2115506 IKZF1 Cell Signaling Technology Cat# 14859S; RRID:AB_2744523 IKZF3 Cell Signaling Technology Cat# 15103S; RRID:AB_2744524 CDK12 (IHC) Sigma-Aldrich Cat# HPA008038-25; RRID:AB_1078570 p-AKT (T308) Cell Signaling Technology Cat# 9275S; RRID:AB_329828 phoGSK2b (s9) Cell Signaling Technology Cat# 9336S; RRID:AB_331405 GSK2b Proteintech Cat# 22104-1-AP; RRID:AB_2878997 FYTTD1 Invitrogen Cat# PA5-98705; RRID:AB_2813318 DDIT3 Proteintech Cat# 15204-1-AP; RRID:AB_2292610 MAPK9 Proteintech Cat# 51153-1-AP; RRID:AB_10898168 CDK9 Cell Signaling Technology Cat# 2316S; RRID:AB_2291505 Vinculin Proteintech Cat# 66305-1-Ig; RRID:AB_2810300 GAPDH Cell Signaling Technology Cat# 3683; RRID:AB_1642205 Tubulin Abcam Cat# ab184577; RRID:AB_3661661 Biological samples WA74 Patient-derived xenografts (PDX) University of Michigan N/A PC310 Patient-derived xenografts (PDX) Erasmus Medical Center, Rotterdam, the Netherlands N/A Chemicals, peptides, and recombinant proteins Uprosertib TargetMol Cat# T6849 MK2206 Selleck Chemicals Cat# S1078 Capivasertib Selleck Chemicals Cat# S8019 Thalidomide Selleck Chemicals Cat# S1193 Carfilzomib Selleck Chemicals Cat# S2853 Bafilomycin Selleck Chemicals Cat# S1413 YJ9069 This paper N/A YJ1206 This paper N/A YJ9068 This paper N/A YJ1078 This paper N/A YJ1090 This paper N/A YJ1094 This paper N/A YJ1114 This paper N/A (Continued on next page) e1 Cell Reports Medicine 5, 101752, October 15, 2024 OPEN ACCESS

Techniques: In Vivo, Standard Deviation, Western Blot, Control, Staining, Immunohistochemistry, TUNEL Assay, Derivative Assay

Journal: Cell reports. Medicine

Article Title: Development of an orally bioavailable CDK12/13 degrader and induction of synthetic lethality with AKT pathway inhibition.

doi: 10.1016/j.xcrm.2024.101752

Figure Lengend Snippet: Figure 5. CDK12/13 degradation induces synthetic lethality in conjunction with AKT pathway inhibition in vitro (A) Human phosphorylation pathway profiling array analysis of VCaP cells treated with YJ1206 (500 nM) for 15 h. (B) The PI3K/AKT signaling pathway. The top phosphorylated proteins identified in Figure 5A are highlighted in red. (C) Immunoblot of the noted proteins in VCaP cells treated with siRNA targeting CDK12 and/or CDK13 or YJ1206 at increasing concentrations and time durations. Tubulin is the loading control probed on all immunoblots. (D) Real-time growth curves of VCaP cells upon treatment with siCDK12/13 and/or uprosertib. Data are presented as mean ± standard deviation from three independent experiments. (E) Cell viability of VCaP cells treated with siCDK12/13 and/or uprosertib by CellTiter-Glo assay (two-sided t test, n = 3 independent experiments). (F and G) Real-time growth curves of 22Rv1 cells upon treatment with YJ1206 and/or uprosertib/capivasertib. Data are presented as mean ± standard deviation from n = 3 independent experiments. (H and I) PC310 PDX organoids were treated with YJ1206 and/or uprosertib/capivasertib at varied concentrations to determine the effect on cell growth and drug synergism, with assessments using the Bliss independence method. Red peaks in the 3D plots denote synergy, and the average synergy scores are noted above the plots. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies CDK12 Proteintech Cat# 26816-1-AP; RRID:AB_2880645 CDK13 Millipore Cat# ABE1860 RNA pol II CTD phospho Ser2 Cell Signaling Technology Cat# 13499S; RRID: RRID: AB_2798238 c-PARP Cell Signaling Technology Cat# 5625S; RRID:AB_10699459 gH2AX Abcam Cat# ab11174; RRID:AB_297813 CCNK Bethyl Laboratories Cat# A301-939A; RRID:AB_1547934 p-AKT (ser473) Cell Signaling Technology Cat# 4060S; RRID:AB_2315049 p-PRAS40 Cell Signaling Technology Cat# 2997S; RRID:AB_2258110 AKT Cell Signaling Technology Cat# 4691S; RRID:AB_915783 PRAS40 Cell Signaling Technology Cat# 2610S; RRID:AB_916206 p-S6 (Ser235/236) Cell Signaling Technology Cat# 2211S; RRID:AB_331679 GSPT1 Proteintech Cat# 10763-1-AP; RRID:AB_2115506 IKZF1 Cell Signaling Technology Cat# 14859S; RRID:AB_2744523 IKZF3 Cell Signaling Technology Cat# 15103S; RRID:AB_2744524 CDK12 (IHC) Sigma-Aldrich Cat# HPA008038-25; RRID:AB_1078570 p-AKT (T308) Cell Signaling Technology Cat# 9275S; RRID:AB_329828 phoGSK2b (s9) Cell Signaling Technology Cat# 9336S; RRID:AB_331405 GSK2b Proteintech Cat# 22104-1-AP; RRID:AB_2878997 FYTTD1 Invitrogen Cat# PA5-98705; RRID:AB_2813318 DDIT3 Proteintech Cat# 15204-1-AP; RRID:AB_2292610 MAPK9 Proteintech Cat# 51153-1-AP; RRID:AB_10898168 CDK9 Cell Signaling Technology Cat# 2316S; RRID:AB_2291505 Vinculin Proteintech Cat# 66305-1-Ig; RRID:AB_2810300 GAPDH Cell Signaling Technology Cat# 3683; RRID:AB_1642205 Tubulin Abcam Cat# ab184577; RRID:AB_3661661 Biological samples WA74 Patient-derived xenografts (PDX) University of Michigan N/A PC310 Patient-derived xenografts (PDX) Erasmus Medical Center, Rotterdam, the Netherlands N/A Chemicals, peptides, and recombinant proteins Uprosertib TargetMol Cat# T6849 MK2206 Selleck Chemicals Cat# S1078 Capivasertib Selleck Chemicals Cat# S8019 Thalidomide Selleck Chemicals Cat# S1193 Carfilzomib Selleck Chemicals Cat# S2853 Bafilomycin Selleck Chemicals Cat# S1413 YJ9069 This paper N/A YJ1206 This paper N/A YJ9068 This paper N/A YJ1078 This paper N/A YJ1090 This paper N/A YJ1094 This paper N/A YJ1114 This paper N/A (Continued on next page) e1 Cell Reports Medicine 5, 101752, October 15, 2024 OPEN ACCESS

Techniques: Inhibition, In Vitro, Phospho-proteomics, Western Blot, Control, Standard Deviation, Glo Assay

Journal: Cell reports. Medicine

Article Title: Development of an orally bioavailable CDK12/13 degrader and induction of synthetic lethality with AKT pathway inhibition.

doi: 10.1016/j.xcrm.2024.101752

Figure Lengend Snippet: Figure 7. Mechanism of action of CDK12/13 degrader-induced AKT phosphorylation in prostate cancer The CDK12/13 degrader inhibits Ser2 phosphorylation on RNAPII, disrupting gene expression and leading to DNA damage and instability. This DNA damage triggers AKT phosphorylation, promoting synthetic lethality with AKT inhibition.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies CDK12 Proteintech Cat# 26816-1-AP; RRID:AB_2880645 CDK13 Millipore Cat# ABE1860 RNA pol II CTD phospho Ser2 Cell Signaling Technology Cat# 13499S; RRID: RRID: AB_2798238 c-PARP Cell Signaling Technology Cat# 5625S; RRID:AB_10699459 gH2AX Abcam Cat# ab11174; RRID:AB_297813 CCNK Bethyl Laboratories Cat# A301-939A; RRID:AB_1547934 p-AKT (ser473) Cell Signaling Technology Cat# 4060S; RRID:AB_2315049 p-PRAS40 Cell Signaling Technology Cat# 2997S; RRID:AB_2258110 AKT Cell Signaling Technology Cat# 4691S; RRID:AB_915783 PRAS40 Cell Signaling Technology Cat# 2610S; RRID:AB_916206 p-S6 (Ser235/236) Cell Signaling Technology Cat# 2211S; RRID:AB_331679 GSPT1 Proteintech Cat# 10763-1-AP; RRID:AB_2115506 IKZF1 Cell Signaling Technology Cat# 14859S; RRID:AB_2744523 IKZF3 Cell Signaling Technology Cat# 15103S; RRID:AB_2744524 CDK12 (IHC) Sigma-Aldrich Cat# HPA008038-25; RRID:AB_1078570 p-AKT (T308) Cell Signaling Technology Cat# 9275S; RRID:AB_329828 phoGSK2b (s9) Cell Signaling Technology Cat# 9336S; RRID:AB_331405 GSK2b Proteintech Cat# 22104-1-AP; RRID:AB_2878997 FYTTD1 Invitrogen Cat# PA5-98705; RRID:AB_2813318 DDIT3 Proteintech Cat# 15204-1-AP; RRID:AB_2292610 MAPK9 Proteintech Cat# 51153-1-AP; RRID:AB_10898168 CDK9 Cell Signaling Technology Cat# 2316S; RRID:AB_2291505 Vinculin Proteintech Cat# 66305-1-Ig; RRID:AB_2810300 GAPDH Cell Signaling Technology Cat# 3683; RRID:AB_1642205 Tubulin Abcam Cat# ab184577; RRID:AB_3661661 Biological samples WA74 Patient-derived xenografts (PDX) University of Michigan N/A PC310 Patient-derived xenografts (PDX) Erasmus Medical Center, Rotterdam, the Netherlands N/A Chemicals, peptides, and recombinant proteins Uprosertib TargetMol Cat# T6849 MK2206 Selleck Chemicals Cat# S1078 Capivasertib Selleck Chemicals Cat# S8019 Thalidomide Selleck Chemicals Cat# S1193 Carfilzomib Selleck Chemicals Cat# S2853 Bafilomycin Selleck Chemicals Cat# S1413 YJ9069 This paper N/A YJ1206 This paper N/A YJ9068 This paper N/A YJ1078 This paper N/A YJ1090 This paper N/A YJ1094 This paper N/A YJ1114 This paper N/A (Continued on next page) e1 Cell Reports Medicine 5, 101752, October 15, 2024 OPEN ACCESS

Techniques: Phospho-proteomics, Gene Expression, Inhibition