Journal: Theranostics

Article Title: S6K1 phosphorylation-dependent degradation of Mxi1 by β-Trcp ubiquitin ligase promotes Myc activation and radioresistance in lung cancer

doi: 10.7150/thno.22552

Figure Lengend Snippet: β-Trcp binds to Mxi1 and promotes the ubiquitination and degradation of Mxi1. A. Mxi1-associated proteins in HEK293T cells identified by tandem affinity purification and mass spectrometry analysis were presented. The recovered peptide numbers for a given protein were listed as indicated. B. Exogenously expressed β-Trcp interacts with Mxi1 and vice versa. HEK293T cells were transfected with indicated constructs. Cell lysates were subjected to pulldown using S protein beads or immunoprecipitation (IP) using anti-Myc beads and then analyzed by Western blotting using indicated antibodies (n=3). C. Endogenous β-Trcp binds to Mxi1 and vice versa. HeLa cell lysates were subjected to immunopreciptation using IgG or anti-β-Trcp antibodies and then analyzed by Western blotting as indicated (n=3). D. β-Trcp knockdown leads to accumulation of endogenous Mxi1. HeLa and H1299 cells were transfected with control or β-Trcp siRNA for 48 h and then analyzed by Western blotting as indicated (n=3). The ratio shows relative Mxi1 protein expression normalized for GAPDH (control, set at 1). E. Overexpression of β-Trcp1 decreases the levels of Mxi1 protein. HeLa cells were transfected with increasing amounts of β-Trcp1, followed by Western blotting with indicated antibodies (n=3). The ratio shows relative Mxi1 protein expression normalized for GAPDH (control, set at 1). F. HeLa cells were transfected with indicated constructs for 24 h and treated with MG132 (10 μM) for another 4 h. The samples were subjected to immunopreciptation (IP) using anti-Flag beads and then analyzed by Western blotting using indicated antibodies (n=3). WCL: whole cell lysate. G. Upper panel: HeLa cells transfected with indicated siRNA for 48 h were treated with CHX (20 mg/mL) for the indicated times and then analyzed by Western blotting using indicated antibodies. Lower panel: Quantification of the Mxi1 band intensities over time (n=3). H. Alignment of the candidate phosphodegron sequence in Mxi1 from different species. I. HeLa cells stably expressing empty vector (EV), wild-type Mxi1, or Mxi1 S160A mutant were transfected with indicated constructs and then treated with MG132 (10 μM) for another 4 h. The samples were analyzed as described in (E) (n=3).

Article Snippet: The following antibodies in our study were used for immunoblotting: Phospho-Mxi1 antibody, which was raised against peptide RIRMDS(p)IGSTISS by immunizing rabbits; anti-Mxi1 antibodies (sc-1042, Santa Cruz Biotechnology and HPA035319, Sigma-Aldrich); anti-S6K1 (sc-230), anti-Myc (sc-40), anti-Cdc20 (sc-13162), and anti-GAPDH (sc-166574) antibodies (Santa Cruz Biotechnology); anti-phospho-S6K1(Thr389) (#9234), anti-β-Trcp1 (#4394), anti-Cyclin B1(#4138), anti-Ubiquitin (#3933) and anti-HA (#3724) antibodies (Cell Signaling Technology); anti-Max (ab199489) , anti-β-Trcp2 (ab137770) and anti-Myc (ab32072) antibodies (Abcam); anti-RSK2 (23762-1-AP) and anti-GAPDH (60004-1-Ig) antibodies (Proteintech); anti-Flag (F1804), anti-Actin (A5441) and anti-Tubulin (T-5168) antibodies (Sigma-Aldrich).

Techniques: Affinity Purification, Mass Spectrometry, Transfection, Construct, Immunoprecipitation, Western Blot, Expressing, Over Expression, Sequencing, Stable Transfection, Plasmid Preparation, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: CD147 confers temozolomide resistance of glioma cells via the regulation of β-TrCP/Nrf2 pathway

doi: 10.7150/ijbs.60894

Figure Lengend Snippet: CD147 dictates Nrf2 stability through the suppression of GSK3β/β-TrCP dependent protein degradation (A-B) The indicated proteins were determined in U251 cells with CD147 knockdown (A) or overexpression (B). (C) Nrf2 and GSK3β protein levels were determined in U251 cells following Capivasertib (10nM) treatment. (D-E) Immunoprecipitation was performed to determine the interaction between β-TrCP and Nrf2 in indicated U251 cells. After the β-TrCP protein was immunoprecipitated with an anti-β-TrCP antibody, indicated proteins were detected by western blotting. (F) Suppresion of CD147 decreased the total and nuclear Nrf2 expression. β-Actin and Lamin B served as internal controls. WCL, whole cell lysate; N, nuclear; C, cytoplasmic.

Article Snippet: The lysate was centrifuged, then incubated with anti-β-TrCP antibody (#4394, Cell signaling Technology) overnight at 4°C.

Techniques: Over Expression, Immunoprecipitation, Western Blot, Expressing

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: lincRNA-p21 Mediates the Anti-Cancer Effect of Ginkgo Biloba Extract EGb 761 by Stabilizing E-Cadherin Protein in Colon Cancer

doi: 10.12659/MSM.911924

Figure Lengend Snippet: lincRNA-p21 blocks BTRC-mediated E-cadherin ubiquitination. ( A ) Western blot analysis of BTRC and E-cadherin level in HCT116 cells transfected with BTRC overexpression vector. ( B ) Western blot analysis of E-cadherin level in HCT116 cells treated with HA-BTRC or lincRNA-p21 in combination, as indicated. ( C ) HCT116 cells were overexpressed with lincRNA-p21 and treated with MG132 for 6 h, then anti-BTRC antibody was used for immunoprecipitation assay.

Article Snippet: Then, the membrane was blocked with 5% (5 g/100 mL) nonfat dry milk in tri-buffered saline plus Tween (TBS-T) buffer for 2 h at room temperature and incubated with specific primary antibodies (1: 1000 solution) at 4°C overnight, followed by horseradish peroxidase-conjugated (HRP) secondary antibodies at room temperature for 1 h. The primary antibodies used were: anti-E-cadherin (Abcam, ab1416), anti-Ubiquitin (Abcam, ab7780) and anti-BTRC (CST, D13F10), FLAG, and HA (Sigma, F1804 and H3663).

Techniques: Western Blot, Transfection, Over Expression, Plasmid Preparation, Immunoprecipitation

Journal: Nature Communications

Article Title: Revealing β-TrCP activity dynamics in live cells with a genetically encoded biosensor

doi: 10.1038/s41467-022-33762-3

Figure Lengend Snippet: a Schematic showing several known substrates of β-TrCP and the pathways it regulates. b β-TrCP recognizes two types of degron sequences. Canonical substrates like Emi1 contain the degron DpSGXXpS whereby the S or serine residue must be phosphorylated to bind to β-TrCP. Non-canonical substrates like CDC25B contain a degron consisting of phospho-mimetic amino acids (D or E) in place of serine residues. For example, CDC25B contains a DDGFVD degron sequence. c Various constructs with fragments of human CDC25B fused to mVenus. Each construct was evaluated using the criteria listed. The construct with the best attributes was NLS-CDC25B (aa198–338)-mVenus (hereafter β-TrCP reporter). NLS nuclear localization signal, C cytoplasmic, N nuclear, √ = Pass, x = Fail. d HeLa cells expressing the β-TrCP reporter and H2B-mTurquoise to visualize the nucleus. Cells were treated as indicated with either β-TrCP1,2 siRNA, transfected with full-length β-TrCP1 (β-TrCP OE) or β-TrCP with the F-box deleted (ΔF-β-TrCP1 OE). Images were taken 48 h after transfection. Scale bar is 10 μm. Representative images from n = 3 experiments. e Histograms depicting β-TrCP reporter levels in single cells from ( d ) N = 2431 (siControl), 18,506 (siβ-TrCP), 16,557 (β-TrCP OE), and 1380 (ΔF-β-TrCP1 OE) cells. f HeLa cells expressing the β-TrCP reporter and H2B-mTurquoise to visualize the nucleus. Cells were treated with MLN-4924 (3 μM), cycloheximide (100 μg/mL), NCS (200 ng/mL), or DMSO for 4 h before capturing the image. Scale bar is 10 μm. Representative images from n = 3 experiments. g Histograms depicting β-TrCP reporter levels in single cells from ( f ) N = 8180 (DMSO), 6749 (MLN-4924), 9118 (Cycloheximide) cells. Source data for all figure panels are provided as a Source Data file.

Article Snippet: The following antibodies were used in this study: anti-mVenus (MyBioSource, MBS448126, 1:1000), anti-β-TrCP (Abcam, ab71753, 1:800), anti-β-TrCP (SCBT, sc390629; used for IPs, 2 µg), anti-β-TrCP (CST, 4394, used for IF, 1:250), anti-β-Catenin (BD-610153, 1:2500), anti-Vinculin (V9131, Sigma, 1:5000), anti-FBXW7 (Abcam, ab109617, 1:800), anti-FLAG (Sigma, F3165, 1:2000), anti-Ubiquitin (SCBT, sc8017, 1:800), anti-Cullin1 (SCBT, sc17775, 1:800), anti-SKP1 (SCBT, sc5281, 1:700), anti-CDC25B (CST, 9525, 1:1000), anti-Emi1 (SCBT, sc365212, 1:800), anti-HSP90 (CST, 4877, 1:1000), anti-Bcl-XL (CST, 2764, 1:1000), anti-Bcl2 (CST, 3498, 1:1000), anti-p65 (CST, 8242, 1:1000), anti-IKBα (CST, 4812, 1:1000), anti-caspase 3 (CST, 14220, 1:1000), anti-Cullin1 (CST, 4995, 1:1000), anti-cleaved PARP (CST, 5625, 1:1000), anti-NEDD8 (CST, 2745, 1:1000), anti-FBXO31 (Bethyl, A302-047A, 1:800), anti-mouse IgG (SCBT, sc2025, used for IPs, 2 µg), anti-DYRK1A (SCBT, sc100376, 1:800), anti-PFKFB3 (MyBioSource, MBS9604769, 1:1000), anti-FBXW5 (MyBioSource, MBS9611762,1:1000), anti-FBXO25 (MyBioSource, MBS3017705, 1:1000), anti-FBXW11 (Thermo scientific, PA5-109715, 1:1000), anti-cMyc (Abcam, ab32072, 1:800), and anti-β-Actin (Abcam, ab6276, 1:5000).

Techniques: Sequencing, Construct, Expressing, Transfection

Journal: Nature Communications

Article Title: Revealing β-TrCP activity dynamics in live cells with a genetically encoded biosensor

doi: 10.1038/s41467-022-33762-3

Figure Lengend Snippet: a HEK-293T cells were transfected as indicated with empty vector, β-TrCP reporter, or mutant β-TrCP reporter. Transfected cells were treated with 3 μM MLN-4924 for the 6 h before collection. Whole-cell extracts were immunoprecipitated with the indicated antibody followed by immunoblotting with the indicated antibody. Representative blot from n = 3 experiments. b HEK-293T cells were transfected as indicated. Cells were treated either with DMSO or MLN-4924 (3 μM) for 6 h before collection. Cells were harvested and whole-cell protein extracts were immunoblotted for indicated antibodies. Representative blot from n = 3 experiments. c HEK-293T cells were transfected as indicated. Cells were harvested 48 h after transfection and whole-cell protein extracts were immunoblotted for the indicated antibodies. Representative blot from n = 3 experiments. d HEK-293T cells were transfected as indicated. Transfected cells were then treated with MG132 (5 µM) for 6 h. Whole-cell protein extracts were immunoprecipitated with Ni-NTA resins and immunoprecipitates were immunoblotted for mVenus. High mass ladder indicates polyubiquitination. Representative blot from n = 3 experiments. e HEK-293T cells were transfected as indicated. Transfected cells were then treated with MG132 (5 µM) for 6 h. and then whole-cell lysate were precipitated with Ni-NTA resins. Precipitates were probed for mVenus antibody. Representative blot from n = 3 experiments. f HeLa cells stably expressing the mVenus-β-TrCP reporter, were transfected with the indicated siRNAs against β-TrCP1,2 or Cullin1. Cells were treated with MG132 (5 μM) for the 6 h before collection. Whole-cell protein extracts were precipitated with Ni-NTA resins and immunoblotted for mVenus antibody. Representative blot from n = 2 experiments. Source data for all figure panels are provided as a Source Data file.

Article Snippet: The following antibodies were used in this study: anti-mVenus (MyBioSource, MBS448126, 1:1000), anti-β-TrCP (Abcam, ab71753, 1:800), anti-β-TrCP (SCBT, sc390629; used for IPs, 2 µg), anti-β-TrCP (CST, 4394, used for IF, 1:250), anti-β-Catenin (BD-610153, 1:2500), anti-Vinculin (V9131, Sigma, 1:5000), anti-FBXW7 (Abcam, ab109617, 1:800), anti-FLAG (Sigma, F3165, 1:2000), anti-Ubiquitin (SCBT, sc8017, 1:800), anti-Cullin1 (SCBT, sc17775, 1:800), anti-SKP1 (SCBT, sc5281, 1:700), anti-CDC25B (CST, 9525, 1:1000), anti-Emi1 (SCBT, sc365212, 1:800), anti-HSP90 (CST, 4877, 1:1000), anti-Bcl-XL (CST, 2764, 1:1000), anti-Bcl2 (CST, 3498, 1:1000), anti-p65 (CST, 8242, 1:1000), anti-IKBα (CST, 4812, 1:1000), anti-caspase 3 (CST, 14220, 1:1000), anti-Cullin1 (CST, 4995, 1:1000), anti-cleaved PARP (CST, 5625, 1:1000), anti-NEDD8 (CST, 2745, 1:1000), anti-FBXO31 (Bethyl, A302-047A, 1:800), anti-mouse IgG (SCBT, sc2025, used for IPs, 2 µg), anti-DYRK1A (SCBT, sc100376, 1:800), anti-PFKFB3 (MyBioSource, MBS9604769, 1:1000), anti-FBXW5 (MyBioSource, MBS9611762,1:1000), anti-FBXO25 (MyBioSource, MBS3017705, 1:1000), anti-FBXW11 (Thermo scientific, PA5-109715, 1:1000), anti-cMyc (Abcam, ab32072, 1:800), and anti-β-Actin (Abcam, ab6276, 1:5000).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Western Blot, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: Revealing β-TrCP activity dynamics in live cells with a genetically encoded biosensor

doi: 10.1038/s41467-022-33762-3

Figure Lengend Snippet: a Image montage of a single-cell expressing the β-TrCP reporter. Images were taken at the indicated time (h) relative to mitosis. Scale bar is 10 μm. b Representative trace of β-TrCP reporter levels in a single HeLa cell. Note the artificial increase in fluorescence intensity during mitosis (black arrow) due to cell rounding and nuclear envelope breakdown. c Single-cell and median β-TrCP reporter levels in HeLa cells aligned to mitosis. N = 263 cells. d Median β-TrCP reporter levels in HeLa cells treated with either siControl, siβ-TrCP1,2, siCullin1, or siFBXW7. e Schematic diagram showing that β-TrCP reporter levels are controlled through a balance of synthesis and degradation. Treatment of either cycloheximide (CHX) or MLN-4924 can inhibit these processes. f , g Median β-TrCP reporter levels in HeLa cells treated with either DMSO, MLN-4924 (3 μM), or cycloheximide (100 μg/mL). Only cells that were treated in either G1 ( f ) or S/G2 phase ( g ) of the cell cycle were analyzed. h , i Single-cell ( h ) and median ( i ) β-TrCP reporter levels in MCF-10A cells after mock or mitogen withdrawal. Only cells that were in S/G2 phase at the time of mitogen withdrawal were analyzed. N = 93 (Mock withdrawal) and 100 (Mitogen withdrawal) cells. j , k Single-cell ( j ) and median ( k ) β-TrCP reporter levels in quiescent MCF-10A cells that were either mock or mitogen stimulated. N = 100 cells per condition. l MCF-10A cells were starved for 48 h to induce quiescence, and then either mock or mitogen stimulated for 2 h before cell lysates were collected. Active complex formation is assessed by relative association of the SCF component Cullin1 and Skp1 with β-TrCP. Representative blot from n = 2 experiments. Source data for all figure panels are provided as a Source Data file.

Article Snippet: The following antibodies were used in this study: anti-mVenus (MyBioSource, MBS448126, 1:1000), anti-β-TrCP (Abcam, ab71753, 1:800), anti-β-TrCP (SCBT, sc390629; used for IPs, 2 µg), anti-β-TrCP (CST, 4394, used for IF, 1:250), anti-β-Catenin (BD-610153, 1:2500), anti-Vinculin (V9131, Sigma, 1:5000), anti-FBXW7 (Abcam, ab109617, 1:800), anti-FLAG (Sigma, F3165, 1:2000), anti-Ubiquitin (SCBT, sc8017, 1:800), anti-Cullin1 (SCBT, sc17775, 1:800), anti-SKP1 (SCBT, sc5281, 1:700), anti-CDC25B (CST, 9525, 1:1000), anti-Emi1 (SCBT, sc365212, 1:800), anti-HSP90 (CST, 4877, 1:1000), anti-Bcl-XL (CST, 2764, 1:1000), anti-Bcl2 (CST, 3498, 1:1000), anti-p65 (CST, 8242, 1:1000), anti-IKBα (CST, 4812, 1:1000), anti-caspase 3 (CST, 14220, 1:1000), anti-Cullin1 (CST, 4995, 1:1000), anti-cleaved PARP (CST, 5625, 1:1000), anti-NEDD8 (CST, 2745, 1:1000), anti-FBXO31 (Bethyl, A302-047A, 1:800), anti-mouse IgG (SCBT, sc2025, used for IPs, 2 µg), anti-DYRK1A (SCBT, sc100376, 1:800), anti-PFKFB3 (MyBioSource, MBS9604769, 1:1000), anti-FBXW5 (MyBioSource, MBS9611762,1:1000), anti-FBXO25 (MyBioSource, MBS3017705, 1:1000), anti-FBXW11 (Thermo scientific, PA5-109715, 1:1000), anti-cMyc (Abcam, ab32072, 1:800), and anti-β-Actin (Abcam, ab6276, 1:5000).

Techniques: Expressing, Fluorescence

Journal: Nature Communications

Article Title: Revealing β-TrCP activity dynamics in live cells with a genetically encoded biosensor

doi: 10.1038/s41467-022-33762-3

Figure Lengend Snippet: a β-TrCP reporter levels (black) and β-TrCP activity (blue) for one MCF-10A cell. b Single-cell and median traces of β-TrCP activity during the cell cycle. Traces were computationally aligned to time of mitosis. N = 194 cells. c Scatter plot showing the single-cell correlation of β-TrCP activity and β-TrCP protein levels. Following live-cell imaging, MCF-10A, MCF7, and MDA-MB-231 cells stably expressing the β-TrCP reporter were fixed and stained for β-TrCP. d Scatter plot showing the mean β-TrCP activity and β-TrCP protein levels from the single-cell data in ( c ) Error bars represent standard deviation. ρ; correlation coefficient. Representative plots from n = 3 experiments. e Relative association of β-TrCP with the SCF components Cullin1 and SKP1 in MCF7 and MDA-MB-231 cells. Whole-cell lysates were immunoprecipitated with anti-β-TrCP. Resulting immunoprecipitates were resolved and stained with indicated antibodies. Representative blot from n = 3 experiments. f Colony formation assay in MCF7 and MDA-MB-231 cells treated with either control shRNA or β-TrCP1,2 shRNA. Representative image from n = 3 experiments. g Bar graph showing the mean percentage of Annexin-V5 positive cells from Supplementary Fig. . Error bars represent SD from n = 5 experiments. P values were calculated using a two-way ANOVA and Sidak’s multiple comparisons test. n.s. not significant ( p = 0.94). h Immunoblotting of NF-kB target genes (Bcl-XL, Bcl2, p65) in MCF7, MDA-MB-231, and MDA-MB-231 cells treated with β-TrCP1,2 siRNA. Representative blot from n = 3 experiments. i Percent growth inhibition of MCF7 and MDA-MB-231 cells, treated with different doses of doxorubicin. Cells were treated with either control siRNA or β-TrCP1,2 siRNA. Error bars represent SEM from n = 3 experiments. j Bar graph showing the mean IC50 of the dose–response curves in ( i ). Error bars represent SD from n = 3 experiments. P values were calculated using a one-way ANOVA. n.s. not significant ( p = 0.69 and p = 0.88 respectively). Source data for all figure panels are provided as a Source Data file.

Article Snippet: The following antibodies were used in this study: anti-mVenus (MyBioSource, MBS448126, 1:1000), anti-β-TrCP (Abcam, ab71753, 1:800), anti-β-TrCP (SCBT, sc390629; used for IPs, 2 µg), anti-β-TrCP (CST, 4394, used for IF, 1:250), anti-β-Catenin (BD-610153, 1:2500), anti-Vinculin (V9131, Sigma, 1:5000), anti-FBXW7 (Abcam, ab109617, 1:800), anti-FLAG (Sigma, F3165, 1:2000), anti-Ubiquitin (SCBT, sc8017, 1:800), anti-Cullin1 (SCBT, sc17775, 1:800), anti-SKP1 (SCBT, sc5281, 1:700), anti-CDC25B (CST, 9525, 1:1000), anti-Emi1 (SCBT, sc365212, 1:800), anti-HSP90 (CST, 4877, 1:1000), anti-Bcl-XL (CST, 2764, 1:1000), anti-Bcl2 (CST, 3498, 1:1000), anti-p65 (CST, 8242, 1:1000), anti-IKBα (CST, 4812, 1:1000), anti-caspase 3 (CST, 14220, 1:1000), anti-Cullin1 (CST, 4995, 1:1000), anti-cleaved PARP (CST, 5625, 1:1000), anti-NEDD8 (CST, 2745, 1:1000), anti-FBXO31 (Bethyl, A302-047A, 1:800), anti-mouse IgG (SCBT, sc2025, used for IPs, 2 µg), anti-DYRK1A (SCBT, sc100376, 1:800), anti-PFKFB3 (MyBioSource, MBS9604769, 1:1000), anti-FBXW5 (MyBioSource, MBS9611762,1:1000), anti-FBXO25 (MyBioSource, MBS3017705, 1:1000), anti-FBXW11 (Thermo scientific, PA5-109715, 1:1000), anti-cMyc (Abcam, ab32072, 1:800), and anti-β-Actin (Abcam, ab6276, 1:5000).

Techniques: Activity Assay, Live Cell Imaging, Stable Transfection, Expressing, Staining, Standard Deviation, Immunoprecipitation, Colony Assay, shRNA, Western Blot, Inhibition

Journal: Nature Communications

Article Title: Revealing β-TrCP activity dynamics in live cells with a genetically encoded biosensor

doi: 10.1038/s41467-022-33762-3

Figure Lengend Snippet: a Schematic showing the β-TrCP reporter fused to NanoLuc luciferase (NLuc). b Relative luciferase activity of MDA-MB-231 cells stably expressing β-TrCP reporter treated with DMSO, MLN-4924, or cycloheximide (CHX) for 6 h. Graph is a box-and-whisker plot where the red line represents the median, the box represents the inter-quartile range, and the whiskers represent 5–95 percentiles. Dots represent individual data points beyond these percentiles. n = 32 replicates. c Workflow of the high-throughput screening (HTS) strategy. MDA-MB-231 cells stably expressing the β-TrCP-NLuc reporter were seeded in 1536-well plates. The initial screen used the MIPE 5.0, and NPC 2.0 libraries comprising over 5000 approved and investigational drugs. After 6 h of treatment, luminescence was measured using a PHERAstar FSX microplate reader (BMG Labtech). d Scatter plot summarizing luminescence of MDA-MB-231 cells expressing β-TrCP-NLuc reporter treated with compounds from MIPE 5.0 and NPC 2.0 library as described in ( c ) Each dot represents the percent activity of each small-molecule compound tested normalized to neutral and negative controls. Shaded regions highlight compounds demonstrating greater than 50% change relative to DMSO-treated controls. e STRING pathway analysis of proteins whose inhibitors were found to activate β-TrCP activity in our screen. Nodes were colored green if they are a receptor-tyrosine kinase (RTK). f Relative reporter levels of MDA-MB-231 cells expressing either β-TrCP-mVenus reporter, mutant β-TrCP reporter, or Skp2-mCherry reporter treated with increasing doses of Sunitinib. Error bars represent SEM from n = 2 repeats. g Single-cell traces of β-TrCP-mVenus reporter levels in MDA-MB-231 cells treated with Sunitinib at the indicated time. Data is median ± 95% confidence intervals. h Exponentially growing MDA-MB-231 cells were treated with Sunitinib (3 μM) for the indicated times. Whole-cell lysates were resolved in SDS-PAGE and immunoblotted for the indicated proteins. Representative blot of n = 3 independent experiments. i Immunoprecipitation of β-TrCP with the SCF complex components Skp1 and Cullin1 in MDA-MB-231 cells treated with either DMSO or Sunitinib. Quantification of enrichment of bands compared to control is shown beneath the relevant bands. Representative blot of n = 3 independent experiments. j CCK-8 assay to determine percent growth inhibition of MDA-MB-231 cells treated with either control siRNA or β-TrCP1,2 siRNA. Cells were treated with different doses of doxorubicin with or without 5 μM sunitinib. Error bars represent SEM from n = 4 experiments. k Model showing relative β-TrCP activity across quiescence (G0) and different cycling phases. l Discovery of RTK signaling as a modulator of active SCF- β-TrCP complex. Sunitinib malate promotes the association of β-TrCP with core SCF complex components. Source data for all figure panels are provided as a Source Data file.

Article Snippet: The following antibodies were used in this study: anti-mVenus (MyBioSource, MBS448126, 1:1000), anti-β-TrCP (Abcam, ab71753, 1:800), anti-β-TrCP (SCBT, sc390629; used for IPs, 2 µg), anti-β-TrCP (CST, 4394, used for IF, 1:250), anti-β-Catenin (BD-610153, 1:2500), anti-Vinculin (V9131, Sigma, 1:5000), anti-FBXW7 (Abcam, ab109617, 1:800), anti-FLAG (Sigma, F3165, 1:2000), anti-Ubiquitin (SCBT, sc8017, 1:800), anti-Cullin1 (SCBT, sc17775, 1:800), anti-SKP1 (SCBT, sc5281, 1:700), anti-CDC25B (CST, 9525, 1:1000), anti-Emi1 (SCBT, sc365212, 1:800), anti-HSP90 (CST, 4877, 1:1000), anti-Bcl-XL (CST, 2764, 1:1000), anti-Bcl2 (CST, 3498, 1:1000), anti-p65 (CST, 8242, 1:1000), anti-IKBα (CST, 4812, 1:1000), anti-caspase 3 (CST, 14220, 1:1000), anti-Cullin1 (CST, 4995, 1:1000), anti-cleaved PARP (CST, 5625, 1:1000), anti-NEDD8 (CST, 2745, 1:1000), anti-FBXO31 (Bethyl, A302-047A, 1:800), anti-mouse IgG (SCBT, sc2025, used for IPs, 2 µg), anti-DYRK1A (SCBT, sc100376, 1:800), anti-PFKFB3 (MyBioSource, MBS9604769, 1:1000), anti-FBXW5 (MyBioSource, MBS9611762,1:1000), anti-FBXO25 (MyBioSource, MBS3017705, 1:1000), anti-FBXW11 (Thermo scientific, PA5-109715, 1:1000), anti-cMyc (Abcam, ab32072, 1:800), and anti-β-Actin (Abcam, ab6276, 1:5000).

Techniques: Luciferase, Activity Assay, Stable Transfection, Expressing, Whisker Assay, High Throughput Screening Assay, Mutagenesis, SDS Page, Immunoprecipitation, CCK-8 Assay, Inhibition