anti-skp2 mouse  (Thermo Fisher)

Journal: Molecules (Basel, Switzerland)

Article Title: L-Arginine Enhances Oral Keratinocyte Proliferation under High-Glucose Conditions via Upregulation of CYP1A1 , SKP2 , and SRSF5 .

doi: 10.3390/molecules28207020

Figure Lengend Snippet: Figure 6. SKP2 in oral keratinocytes is upregulated after L-Arginine treatment under high-glucose conditions. TIGK cells were treated with 500 µM Arg in a culture medium having high glucose (48 mM) for 24 and 48 h. Cells were fixed in formalin and permeabilized with Triton X-100 followed by incubation with a mouse anti–human SKP2 antibody and a goat anti-mouse IgG Alexa Fluor 488 secondary antibody. Alexa Fluor 594 Phalloidin was used to stain F-actin as a counterstain. DAPI was used for nuclei staining. The green fluorescence intensity of SKP2 was quantified using ImageJ. Scale bar: 50 µm. # p < 0.01 compared to control.

Article Snippet: After 12 h, cells were treated with 500 μM Arg under a high-glucose condition (48 mM) for either 24 or 48 h, after which cells were then fixed in formalin for 10 min. After two washes with PBS, cells were permeabilized with 0.15% Triton X 100 for 10 min and blocked with 5% goat serum in PBS for 1 h at room temperature and subsequently incubated with mouse anti–human CYP1A1 (1A3-03) (Santa Cruz Biotechnology INC, Dallas, TX, USA, Catalog No: sc-101828, 1/100 dilution), mouse anti-human SKP2 (A-2) (Santa Cruz Biotechnology INC, Catalog No: sc-74477, 1/100 dilution), or rabbit anti-human SKP2 (Proteintech, Rosemont, IL, USA, Catalog No: 16237-1-AP, 1/250) overnight at 4 ◦C.

Techniques: Incubation, Staining, Control

Journal: Molecules (Basel, Switzerland)

Article Title: L-Arginine Enhances Oral Keratinocyte Proliferation under High-Glucose Conditions via Upregulation of CYP1A1 , SKP2 , and SRSF5 .

doi: 10.3390/molecules28207020

Figure Lengend Snippet: Figure 7. SKP2 in oral keratinocytes is upregulated after L-Arginine treatment under high-glucose conditions. TIGK cells were treated with 500 µM Arg in a culture medium having high glucose (48 mM) for 24 and 48 h. Cells were fixed in formalin and permeabilized with Triton X-100 followed by incubation with a rabbit anti-human SKP2 antibody and a goat anti-rabbit IgG Alexa Fluor 488 secondary antibody. Alexa Fluor 594 Phalloidin was used to stain F-actin as a counterstain. DAPI was used for nuclei staining. The green fluorescence intensity of SKP2 was quantified using ImageJ. Scale bar: 50 µm. * p < 0.05 compared to control.

Article Snippet: After 12 h, cells were treated with 500 μM Arg under a high-glucose condition (48 mM) for either 24 or 48 h, after which cells were then fixed in formalin for 10 min. After two washes with PBS, cells were permeabilized with 0.15% Triton X 100 for 10 min and blocked with 5% goat serum in PBS for 1 h at room temperature and subsequently incubated with mouse anti–human CYP1A1 (1A3-03) (Santa Cruz Biotechnology INC, Dallas, TX, USA, Catalog No: sc-101828, 1/100 dilution), mouse anti-human SKP2 (A-2) (Santa Cruz Biotechnology INC, Catalog No: sc-74477, 1/100 dilution), or rabbit anti-human SKP2 (Proteintech, Rosemont, IL, USA, Catalog No: 16237-1-AP, 1/250) overnight at 4 ◦C.

Techniques: Incubation, Staining, Control

Journal: Molecules (Basel, Switzerland)

Article Title: L-Arginine Enhances Oral Keratinocyte Proliferation under High-Glucose Conditions via Upregulation of CYP1A1 , SKP2 , and SRSF5 .

doi: 10.3390/molecules28207020

Figure Lengend Snippet: Figure 8. Knocking down CYP1A1, SKP2, or SRSF5 abolishes enhanced oral keratinocyte proliferation induced by L-Arginine treatment under high-glucose conditions. TIGK cells were transfected with 20 nM siRNAs of CYP1A1, SKP2, or SRSF5 and then treated with 500 µM Arg in a culture medium with high glucose (48 mM). (A) mRNA expression of CYP1A1, SKP2, or SRSF5 48 h after transfection. (B) The proliferation of TIGK cells after knocking down CYP1A1, SKP2, or SRSF5 assessed by cell count at 48 h and MTS assay at 72 and 120 h after transfection. (C) Representative images of cell density at 48 h after treatment. Scale bar: 100 µm. * p < 0.05, # p < 0.01.

Article Snippet: After 12 h, cells were treated with 500 μM Arg under a high-glucose condition (48 mM) for either 24 or 48 h, after which cells were then fixed in formalin for 10 min. After two washes with PBS, cells were permeabilized with 0.15% Triton X 100 for 10 min and blocked with 5% goat serum in PBS for 1 h at room temperature and subsequently incubated with mouse anti–human CYP1A1 (1A3-03) (Santa Cruz Biotechnology INC, Dallas, TX, USA, Catalog No: sc-101828, 1/100 dilution), mouse anti-human SKP2 (A-2) (Santa Cruz Biotechnology INC, Catalog No: sc-74477, 1/100 dilution), or rabbit anti-human SKP2 (Proteintech, Rosemont, IL, USA, Catalog No: 16237-1-AP, 1/250) overnight at 4 ◦C.

Techniques: Transfection, Expressing, Cell Counting, MTS Assay

Journal: Molecular Oncology

Article Title: Lysosome‐dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer

doi: 10.1002/1878-0261.13497

Figure Lengend Snippet: Enhanced SKP2:FOXA1 interaction correlates with advanced PCa in human specimens. (A, B) Immunofluorescence (IF) images demonstrate the colocalization of SKP2 (green) and FOXA1 (red) proteins in primary prostate tumors. White arrows represent SKP2 and FOXA1 protein colocalization. Scale bars are 10 μm. (C) Immunohistochemistry (IHC) staining for SKP2 and FOXA1 in tissue microarray (TMA) of primary prostate tumors ( n = 80). The representative images shown are H&E, SKP2, and FOXA1. Black arrows represent high SKP2 and low FOXA1 protein levels. Red arrows represent low SKP2 and high FOXA1 protein. Scale bars are 100 μm. (D) Intensity scores for SKP2 and FOXA1 staining were graded as 0, 1, 2, and 3 and the respective statistical significance was determined by Chi‐square test (Fig. ) and Pearson correlation coefficient. (E) SKP2‐to‐FOXA1 ratio (SKP2:FOXA1) for IHC staining for SKP2 and FOXA1 TMA of primary prostate tumors ( n = 45). (F, G) SKP2 mRNA expression is elevated in recurrent PCa (GSE25136; P = 0.0459) and neuroendocrine PCa (NEPC) ( P = 0.0224). CRPC refers to Castration‐resistant PCa. Previously published PCa gene expression datasets were retrieved from GEO database ( ncbi.nlm.nih.gov/gds /) and cbioportal ( cbioportal.org ), respectively. The corresponding normalized SKP2 levels were plotted. (H) Immunoblot analysis for several PCa cell lines. Quantification analysis of the relative protein levels for SKP2 and SYP is displayed to the right ( n = 4). Comparison between groups was performed using Student's t ‐test. Bars indicate SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then incubated with primary antibodies including mouse anti‐SKP2 (2 μg·mL −1 , Invitrogen, 32‐3300), rabbit anti‐FOXA1 (1 : 250, Abcam, Cambridge, UK, ab170933), rat anti‐LAMP2 (1 : 1000, Invitrogen, MA1‐165), and mouse anti‐Myc tag (5 μg·mL −1 , Abcam, ab32) as previously described [ ].

Techniques: Immunofluorescence, Immunohistochemistry, Microarray, Staining, Expressing, Western Blot, Comparison

Journal: Molecular Oncology

Article Title: Lysosome‐dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer

doi: 10.1002/1878-0261.13497

Figure Lengend Snippet: SKP2 regulates FOXA1 through K6 and K29‐linked ubiquitination. (A, B) Immunoblot analysis displays FOXA1 elevation in C4‐2B ( n = 3) and 22Rv1 ( n = 3) cells upon SKP2 knockdown (KD) via shRNA. Quantification analysis of the relative protein levels for SKP2 and FOXA1 is displayed on the right. (C) Immunofluorescence (IF) images demonstrate the colocalization of endogenous SKP2 and FOXA1 proteins in C4‐2B and 22Rv1 PCa cells. Scale bars are 20 μm. (D, E) Co‐immunoprecipitation analysis displays a physical interaction for SKP2 and FOXA1 proteins in HEK293T ( n = 3) cells using Myc‐tagged SKP2 or Flag‐tagged FOXA1. WCL indicates the whole cell lysates. (F, G) Immunoprecipitation analysis displays an interaction for endogenous SKP2 and FOXA1 proteins in C4‐2B ( n = 3) and 22Rv1 ( n = 3) PCa cells. (H) In vivo ubiquitination assay displays an increase in HA‐Ub‐linked ubiquitination for FOXA1 by Myc‐SKP2 in HEK293T ( n = 3) cells. (I) In vivo ubiquitination assay demonstrates a reduction in ubiquitination for K6R and K29R mutants ( n = 3). (J) HEK293T cells were co‐transfected with Flag‐tagged FOXA1, HA‐ubiquitin (wild type, WT), and WT‐Myc‐SKP2 or a ▵F SKP2 mutant before being treated with Cycloheximide (CHX; 100 μg·mL −1 ) protein synthesis inhibitor for the indicated time points (h, hours). EV refers to empty vector. Lower panel is the corresponding plot for FOXA1 protein intensity ( n = 3). (K) The percent and mean fluorescence intensity (MFI) of FOXA1 ubiquitination in HEK293T cells after overexpression of Myc‐SKP2, Flag‐FOXA1, and HA‐Ubiquitin WT or mutants. Colored points represent number of replicates per group. Comparisons between groups were analyzed using paired two‐tailed Student's t ‐test. Bars indicate SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then incubated with primary antibodies including mouse anti‐SKP2 (2 μg·mL −1 , Invitrogen, 32‐3300), rabbit anti‐FOXA1 (1 : 250, Abcam, Cambridge, UK, ab170933), rat anti‐LAMP2 (1 : 1000, Invitrogen, MA1‐165), and mouse anti‐Myc tag (5 μg·mL −1 , Abcam, ab32) as previously described [ ].

Techniques: Western Blot, shRNA, Immunofluorescence, Immunoprecipitation, In Vivo, Ubiquitin Assay, Transfection, Mutagenesis, Plasmid Preparation, Fluorescence, Over Expression, Two Tailed Test

Journal: Molecular Oncology

Article Title: Lysosome‐dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer

doi: 10.1002/1878-0261.13497

Figure Lengend Snippet: Skp2 Abrogation elevates Foxa1 levels in vivo in mouse models. (A) Immunoblot analysis shows the effects of Skp2 on Foxa1 protein levels in mouse embryonic fibroblasts (MEFs) with indicated genotypes. Quantification of protein levels is relative to beta‐Actin and displayed in the right panel ( n = 3). (B, C) Immunofluorescence (IF) images show colocalization among Skp2, Foxa1, and ubiquitin in murine prostate organoids ( n = 3). White arrows indicate colocalization for Skp2, Foxa1, and Ubiquitin. Scale bars are 25 μm. (D) Western blotting analysis of Skp2 and Foxa1 protein levels for anterior prostate (AP) tissues of mice with the indicated genotypes. Quantification analysis for the corresponding protein expression is shown in the right panel ( n = 3). (E) Immunohistochemistry (IHC) staining of Skp2 and Foxa1 in prostate tissues of mice with the indicated genotypes. The side panel displays a quantification analysis for IHC staining ( n = 3). Scale bars are 100 μm. (F) IF images show colocalization (white arrows) among Skp2, Foxa1, and Pcna (proliferating cell nuclear antigen) prostate tissues in Pten pc−/− ; Trp53 pc−/− mutant mice, while Skp2 loss in Pten pc−/− ; Trp53 pc−/− ; Skp2 −/− mutant mice has decreased Skp2, Foxa1, and Pcna colocalization ( n = 3). Scale bars are 25 μm. (G) IF images for luminal (CD24 + ) and basal (CD49f + ) lineage markers in Pten pc−/− ; Trp53 pc−/− and Pten pc−/− ; Trp53 pc−/− ; Skp2 −/− mutant mice ( n = 3). Scale bars are 25 μm. Comparison between groups was performed using Student's t ‐test. Bars indicate SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then incubated with primary antibodies including mouse anti‐SKP2 (2 μg·mL −1 , Invitrogen, 32‐3300), rabbit anti‐FOXA1 (1 : 250, Abcam, Cambridge, UK, ab170933), rat anti‐LAMP2 (1 : 1000, Invitrogen, MA1‐165), and mouse anti‐Myc tag (5 μg·mL −1 , Abcam, ab32) as previously described [ ].

Techniques: In Vivo, Western Blot, Immunofluorescence, Expressing, Immunohistochemistry, Mutagenesis, Comparison

Journal: Molecular Oncology

Article Title: Lysosome‐dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer

doi: 10.1002/1878-0261.13497

Figure Lengend Snippet: SKP2 alters FOXA1 ubiquitination levels in human prostate cancer cells in a dose‐dependent manner. (A) Endogenous ubiquitination of FOXA1 in C4‐2B proceeding SKP2 inhibition using SZL P1‐41 at different concentrations (μ m ). Samples were collected and subjected to in vivo ubiquitination and western blot analysis ( n = 3). (B) FOXA1 ubiquitination profile using flow cytometry for vehicle, SKP2 KD, and SKP2 inhibition using SZL P1‐41 for C4‐2B. Representative histogram and percent ubiquitination of FOXA1 are plotted. Colored points represent number of replicates per group. (C) C4‐2B xenograft mice received vehicle (DMSO) or SKP2 inhibitor SZL P1‐41 (30 mg·kg −1 ; three times per week, intraperitoneal, i.p. injection) for 30 days. Immunofluorescence (IF) staining displays increased colocalization (white arrows) amongst SKP2, FOXA1, and PCNA for C4‐2B xenograft vehicle tissues, while exposure to SZL P1‐41 treatment reduced SKP2 and PCNA levels ( n = 3). Scale bars are 25 μm. (D) C4‐2B xenograft tissue sections were subjected to Immunohistochemistry (IHC) staining with the indicated antibodies ( n = 5). Scale bars are 100 μm. (E) IF luminal (CD24 + ) and basal (CD49f + ) lineage staining in C4‐2B vehicle and SZL P1‐41‐treated mice ( n = 3). Scale bars are 25 μm. Comparison between groups was performed using Student's t ‐test. Bars indicate SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then incubated with primary antibodies including mouse anti‐SKP2 (2 μg·mL −1 , Invitrogen, 32‐3300), rabbit anti‐FOXA1 (1 : 250, Abcam, Cambridge, UK, ab170933), rat anti‐LAMP2 (1 : 1000, Invitrogen, MA1‐165), and mouse anti‐Myc tag (5 μg·mL −1 , Abcam, ab32) as previously described [ ].

Techniques: Inhibition, In Vivo, Western Blot, Flow Cytometry, Injection, Immunofluorescence, Staining, Immunohistochemistry, Comparison

Journal: Molecular Oncology

Article Title: Lysosome‐dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer

doi: 10.1002/1878-0261.13497

Figure Lengend Snippet: FOXA1 Regulation by SKP2 is a lysosomal‐dependent event. (A) The effects of Chloroquine on FOXA1 levels in C4‐2B cells. Cells were subjected to MG132 or chloroquine treatment at the indicated concentrations (μ m ). Lower panels display the quantification for FOXA1 protein levels ( n = 3). (B) The effects of bafilomycin A1 (BA1) treatment at the indicated concentrations (μ m ) on FOXA1 protein levels in C4‐2B and 22Rv1 cells. Quantification for FOXA1 levels is displayed below ( n = 3). (C) Immunoblot demonstrating the effects of lysosomal marker (LC3, LAMP1, and LAMP2) KD on FOXA1 protein levels in C4‐2B and 22Rv1 cells. Lower panels demonstrate quantified FOXA1 protein levels ( n = 3). (D) Immunofluorescence (IF) images display an increase in LAMP2 (lysosome‐associated membrane glycoprotein) levels in C4‐2B cells upon Myc‐SKP2 overexpression ( n = 3). Scale bars are 20 μm. (E) CellLight Lysosome‐GFP, BacMam 2.0, labeling of lysosomal activity in association with FOXA1 in C4‐2B ( n = 3) and 22Rv1 ( n = 3). White arrows indicate colocalization between the lysosome and FOXA1. Scale bars are 25 μm. (F) IF images show the impact of Skp2 restoration on Lamp2 in Pten/Trp53/Skp2 triple‐null mouse embryonic fibroblasts (MEFs) proceeding transient overexpression of Myc‐SKP2 ( n = 3). White arrows indicate colocalization amongst Lamp2, FOXA1, and Skp2. Scale bars are 25 μm. (G) qRT‐PCR analysis of Skp2 and the lysosomal genes Hexb and Mcoln1 in Pten/Trp53/Skp2 triple‐null MEFs proceeding the transient overexpression of Myc‐SKP2 or an empty vector (EV). Results represent relative expression values to the housekeeping gene beta‐Actin ( n = 3). (H) Western blotting analysis of Skp2, FOXA1, and Lamp2 levels for Pten/Trp53/Skp2 triple‐null MEFs proceeding overexpression of Myc‐SKP2 (0–2 μg). Quantification analysis for the corresponding relative protein levels is shown in the right panel ( n = 3). (I) A working model on the K6‐ and K29‐linked ubiquitination of FOXA1 by SKP2 in lysosomal degradation. Comparison between groups was performed using Student's t ‐test. Bars indicate SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then incubated with primary antibodies including mouse anti‐SKP2 (2 μg·mL −1 , Invitrogen, 32‐3300), rabbit anti‐FOXA1 (1 : 250, Abcam, Cambridge, UK, ab170933), rat anti‐LAMP2 (1 : 1000, Invitrogen, MA1‐165), and mouse anti‐Myc tag (5 μg·mL −1 , Abcam, ab32) as previously described [ ].

Techniques: Western Blot, Marker, Immunofluorescence, Membrane, Over Expression, Labeling, Activity Assay, Quantitative RT-PCR, Plasmid Preparation, Expressing, Comparison

Journal: PLoS ONE

Article Title: Estrogen and Progesterone Regulate p27kip1 Levels via the Ubiquitin-Proteasome System: Pathogenic and Therapeutic Implications for Endometrial Cancer

doi: 10.1371/journal.pone.0046072

Figure Lengend Snippet: Details are in and each Figure. A, E2 dose-response: ECC-1 and KLE cells were treated with E2 and p27, Skp2, and Cks1 protein levels determined by immunoblotting. B, E2 time course: ECC-1 cells were treated with E2 for the times indicated and protein levels determined as above. C, Pg dose-response: ECC-1 cells were treated with Pg/E2 and p27, Skp2 and Cks1 protein levels determined as above. D, Pg time course: ECC-1 cells were treated with Pg/E2 for the times shown and protein levels as above. Densitometric scans of all protein bands are shown by the graphs to the right of each blot. Each band was normalized to actin and then compared with 0 time or untreated control. The data are expressed as relative intensity of each band ± standard deviation. E, E2/Pg cycloheximide (CHX) treatment: ECC-1 cells were treated with E2 for 18 h or with E2 and Pg for 48 h. CHX was added 6 h prior to final harvest. Cells were harvested at 0,1,2, and 6 h time points. CHX alone was the baseline control. Lysates were prepared and immunoblotted for p27 and Skp2 and the levels of each protein band determined by densitometry (band intensity). The percent change within each treatment parameter was calculated based on the zero time point for each group. Protein turn-over was determined by comparison of cells treated with CHX alone compared to CHX in the presence of each hormone at each time point. The line graphs represent the relative intensity of each band normalized to actin for each group. F, G: E2 stimulates and Pg inhibits proliferation: ECC-1 cells were treated with E2 or Pg/E2 and cell proliferation determined by MTS assay *p<0.05. H, Cell cycle distribution: ECC-1 cells were treated with E2 or E2/Pg and cell cycle analysis performed as described .

Article Snippet: Primary antibodies included: mouse anti-human p27 Kip1 (1∶1000, Clone 57, BD Transduction Laboratories), rabbit anti phospho-p27 (1∶1000, PT187, Invitrogen), mouse anti-human p45/Skp2 (1∶1000, 8D9, Invitrogen), rabbit anti-human Cks1 (1∶500, C-term, Invitrogen), and mouse anti-human Cdh1 (1∶1000, DH01, Calbiochem).

Techniques: Western Blot, Standard Deviation, MTS Assay, Cell Cycle Assay

Journal: PLoS ONE

Article Title: Estrogen and Progesterone Regulate p27kip1 Levels via the Ubiquitin-Proteasome System: Pathogenic and Therapeutic Implications for Endometrial Cancer

doi: 10.1371/journal.pone.0046072

Figure Lengend Snippet: A, B, E2, Skp2 knockdown: ECC-1 cells were transiently transfected with Skp2 or control siRNA, treated with E2 alone or in the presence of 1 µM Lactacystin (Lac) or 10 nM ICI, subjected to cell fractionation, p27 and Skp2 levels determined by immunoblotting, as described in . In Panel A, knock-down efficiency was determined by comparing cell lysates from Skp2 siRNA and control siRNA treated cells by immunoblotting. Relative p27 levels are represented by the densitometric scan below the blot. E2, cell proliferation in Skp2 knockdown cells: ECC-1 cells, transfected with Skp2 or control siRNA, were treated with E2 or not treated and proliferation determined by MTS assay, as described in . Data are presented as an average of two independent experiments. *p≤0.05.

Article Snippet: Primary antibodies included: mouse anti-human p27 Kip1 (1∶1000, Clone 57, BD Transduction Laboratories), rabbit anti phospho-p27 (1∶1000, PT187, Invitrogen), mouse anti-human p45/Skp2 (1∶1000, 8D9, Invitrogen), rabbit anti-human Cks1 (1∶500, C-term, Invitrogen), and mouse anti-human Cdh1 (1∶1000, DH01, Calbiochem).

Techniques: Transfection, Cell Fractionation, Western Blot, MTS Assay

Journal: PLoS ONE

Article Title: Estrogen and Progesterone Regulate p27kip1 Levels via the Ubiquitin-Proteasome System: Pathogenic and Therapeutic Implications for Endometrial Cancer

doi: 10.1371/journal.pone.0046072

Figure Lengend Snippet: A, B, Time course of E2 and Pg [plus E2] treatment of EEC-1 cells. ECC-1 cells were treated with E2 and Pg plus E2, the experiments terminated at the times indicated, lysates prepared, and Cdh1 protein levels determined by immunoblotting, as described in . C, D, E2, Pg, mRNA levels of p27, Skp2, Cks1 and Cdh1: ECC-1 cells were treated with E2 or Pg/E2, total RNA extracted at the times shown, and quantitative real-time RT-PCR performed, all in . PR and glycodelin primers were used as controls for ER and PR target genes, respectively. Data are presented as an average of two independent experiments.

Article Snippet: Primary antibodies included: mouse anti-human p27 Kip1 (1∶1000, Clone 57, BD Transduction Laboratories), rabbit anti phospho-p27 (1∶1000, PT187, Invitrogen), mouse anti-human p45/Skp2 (1∶1000, 8D9, Invitrogen), rabbit anti-human Cks1 (1∶500, C-term, Invitrogen), and mouse anti-human Cdh1 (1∶1000, DH01, Calbiochem).

Techniques: Western Blot, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Estrogen and Progesterone Regulate p27kip1 Levels via the Ubiquitin-Proteasome System: Pathogenic and Therapeutic Implications for Endometrial Cancer

doi: 10.1371/journal.pone.0046072

Figure Lengend Snippet: A, E2; B, Pg, Cell fractionation and analysis of Cdh1, Skp2, Cks1, p27 proteins and p-p27(T187): Cells were either untreated, treated with E2, E2 plus Lac or treated with Pg/E2 with or without 1 µM Lactacystin (Lac) or RU486. Cells were fractionated and proteins analyzed by immunoblotting, all described in . The inset in A shows that lactacystin decreases Skp2 protein levels. C., Pg, Cdh1 binding to APC: Cells were treated with Pg/E2, cell lysates immunoprecipitated with anti-Cdh1 followed by immunoblotting with anti-Cdc27, as described in . D, diagram depicts APC/Cdh1 as the upstream E3 ligase that ubiquitylates Skp2/Cks1 (of the SCF complex), which is the downstream E3 ligase that ubiquitylates p27 to regulate p27 protein levels.

Article Snippet: Primary antibodies included: mouse anti-human p27 Kip1 (1∶1000, Clone 57, BD Transduction Laboratories), rabbit anti phospho-p27 (1∶1000, PT187, Invitrogen), mouse anti-human p45/Skp2 (1∶1000, 8D9, Invitrogen), rabbit anti-human Cks1 (1∶500, C-term, Invitrogen), and mouse anti-human Cdh1 (1∶1000, DH01, Calbiochem).

Techniques: Cell Fractionation, Western Blot, Binding Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Estrogen and Progesterone Regulate p27kip1 Levels via the Ubiquitin-Proteasome System: Pathogenic and Therapeutic Implications for Endometrial Cancer

doi: 10.1371/journal.pone.0046072

Figure Lengend Snippet: Cdc14 phosphatase keeps Cdh1 bound to APC; this is the E3 ligase that ubiquitylates Skp2/Cks1 of the SCF complex. E2 decreases nuclear [APC]Cdh1 and induces phosphorylation of p27 on T187 signaling its ubiquitylation by [SCF]-Skp2/Cks1. The decrease in [APC]Cdh1 prevents ubiquitin-mediated degradation of Skp2/Cks1 allowing [SCF]Skp2/Cks1 to ubiquitylate p27 causing its proteasomal degradation; cells progress through S phase. Pg increases [APC]Cdh1, which ubiquitylates [SCF]Skp2/Cks1 causing their degradation blocking p27 degradation; cells are blocked in G1. Ovals are E3 ligases; green small squares are chains of ubiquitin (Ub); small yellow circles indicate phosphorylation (P). Center Table: Slight (<15%); v* = variable, slightly up in ECC-1 and variable EECs. Cdh1 was not present in the cytoplasm in ECC-1 cells or EECs but was found in primary ECA cells (n = 2/3).

Article Snippet: Primary antibodies included: mouse anti-human p27 Kip1 (1∶1000, Clone 57, BD Transduction Laboratories), rabbit anti phospho-p27 (1∶1000, PT187, Invitrogen), mouse anti-human p45/Skp2 (1∶1000, 8D9, Invitrogen), rabbit anti-human Cks1 (1∶500, C-term, Invitrogen), and mouse anti-human Cdh1 (1∶1000, DH01, Calbiochem).

Techniques: Blocking Assay