Journal: Cancers

Article Title: miR-1227 Targets SEC23A to Regulate the Shedding of Large Extracellular Vesicles

doi: 10.3390/cancers13225850

Figure Lengend Snippet: MiR-1227 induces shedding of L-EV and inhibits the shedding of S-EV in PC cells. ( A ) MiR-1227 (teal) and miR-21 (grey) expression in different biological fluids from Weber et al. 2010 . ( B ) Taqman qPCR quantification of miR-1227 in EV isolated from pooled normal or PC patient plasma ( n = 1). ( C ) Transwell migration of RWPE-2 PC and U87 glioma cells stably expressing miR-1227 (teal) or empty vector control (grey) ( n = 3) normalized to corresponding vector control; ( D ) qPCR for miR-1227 from S-EV (blue) and L-EV (red) isolated from PC3 cells stably expressing miR-1227( n = 1). ( E , F ) Digital droplet qPCR quantification of miR-1227 per S-EV ( E ) and L-EV ( F ) ( n = 3). ( G , H ) Atomic force microscopy quantifications of S-EV ( G ) and L-EV ( H ) shed from PC3 cells stably expressing miR-1227 ( n = 3). ( I – L ) Quantification of S-EV ( I ) and L-EV ( J ) shed from PC3 cells stably expressing miR-1227 by TRPS with size distribution plots in ( K , L ), respectively ( n = 3); * p < 0.05.

Article Snippet: PC3 (prostate cancer, human), RWPE-2 (prostate cancer, human), and U87 (glioma, human) cells were purchased from ATCC.

Techniques: Expressing, Isolation, Clinical Proteomics, Migration, Stable Transfection, Plasmid Preparation, Control, Microscopy

Journal: Cancers

Article Title: miR-1227 Targets SEC23A to Regulate the Shedding of Large Extracellular Vesicles

doi: 10.3390/cancers13225850

Figure Lengend Snippet: SEC23A is a direct target of miR-1227; ( A ) qPCR for high confidence miR-1227 target genes involved in vesicle formation from PC3 cells stably expressing miR-1227 ( n = 3). ( B ) Western blot validation of SEC23A reduced expression in PC3, RWPE-2, and U87 cells stably expressing miR-1227. ( C ) Prostate cancer transcriptome atlas analysis of SEC23A in prostate cancer, showing reduced expression levels of SEC23A in cancer versus benign prostate tissue and in metastatic castration resistant versus primary prostate cancer. ( D – F ) Luciferase assays from RWPE-2 cells transfected with miR-1227 or control miRNA, plus the SEC23A 3′UTR luciferase construct ( D ) or the SEC23A 3′UTR luciferase construct with the deletion of 2 different predicted miR-1227 binding sites ( E , F ) ( n = 3); * p < 0.05; ** p < 0.005.

Article Snippet: PC3 (prostate cancer, human), RWPE-2 (prostate cancer, human), and U87 (glioma, human) cells were purchased from ATCC.

Techniques: Stable Transfection, Expressing, Western Blot, Biomarker Discovery, Luciferase, Transfection, Control, Construct, Binding Assay

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Correlation between CPN1 and RAI14. (A) Co-immunoprecipitation (Co-IP) in 293T cells with CPN1 overexpression. (B) A total of 29 differential proteins with more than identified five peptides in immunoprecipitation. (C) Validation of CPN1 interacting with RAI14.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Over Expression, Biomarker Discovery

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: High expression of RAI14 in breast cancer. (A) Expression levels of human RAI14 in different tumor types from TCGA database. (B) The mRNA expression of RAI14 in breast cancer and normal tissues from GEPIA. (C) The protein expression of RAI14 in breast cancer and normal tissues from UALCAN database. (D) High expression of RAI14 in human breast cancer from Human Protein Atlas (HPA) database. * p < 0.05, *** p < 0.001.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Correlation analysis of RAI14 expression levels with pathological type parameters of breast cancer. (A) The mRNA levels of RAI14 in mammary carcinoma with different molecular types. (B) Protein levels of RAI14 in mammary carcinoma with different molecular types. (C) Expression of RAI14 in different mammary carcinoma stages. (D) RAI14 levels in different menopausal states. * p < 0.05, *** p < 0.001.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Relationship between mRNA expression of RAI14 and clinicopathological parameters of breast cancer.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Expressing, Immunohistochemistry, Sequencing

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Differential expression of RAI14 in mammary carcinoma specimens with different clinicopathological parameters from BC-GenExMiner v4.4 data analysis. (A–C) Receptor status (ER + vs. ER-, PR + vs. PR-, HER2+ vs. HER2-). (D) The differential expression of RAI14 in the TNBC and non-TNBC. (E–I) The differential expression of (E) wild-type and mutant, (F) age (≤51 and >51 years), (G) molecular types, (H) clinical tumor stage (I, II, III, IV), and (I) clinicopathological type (IDC, ILC).

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Quantitative Proteomics, Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Kaplan–Meier survival curves of RAI14 in different breast cancer molecular subtypes. (A, B) Survival curves for overall survival (OS) and recurrence-free survival (RFS) in triple-negative breast cancer (n = 1494). (C, D) Survival curves for OS and RFS in luminal B breast cancer (n = 2015). (E, F) Survival curves for OS and RFS in luminal A breast cancer (n = 3511). (G, H) Survival curves for OS and RFS in HER2+ breast cancer for OS and RFS (n = 515).

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Correlation analysis between RAI14 and related genes and markers of immune cells in breast cancer.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: Correlation of RAI14 levels with immune infiltration in TNBC. (A) The expression level of RAI14 was positively correlated with programmed cell death-ligand 1 (CD274) and programmed cell death protein 1(PDCD1). X-axis represents the expression level of immune cell gene markers, and Y-axis represents RAI14. Log2TPM that indicates gene expression level. (B) The expression of immunohistochemical (IHC)staining of CPN1, RAI14, and PDL1 in patients with TNBC tissues. (C) Correlation among IHC scores of CPN1, RAI14, and PDL1.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Immunohistochemistry

Journal: Frontiers in Pharmacology

Article Title: High Expression of RAI14 in Triple-Negative Breast Cancer Participates in Immune Recruitment and Implies Poor Prognosis Through Bioinformatics Analyses

doi: 10.3389/fphar.2022.809454

Figure Lengend Snippet: In TNBC cell lines, CPN1 positively correlated with RAI14 and PDL1 expression. (A) Expression levels of CPN1, RAI14, and PDL1 in breast cancer cell lines. (B) Increased expression of RAI14 and PDL1 in MDA-MB-231 cell lines with overexpression of CPN1. (C) Our protocol summarized in a schematic diagram.

Article Snippet: In brief, the following primary antibodies were used: CPN1 antibody (Abcam, Cambridge, MA, United States, ab232802), RAI14 antibody (Proteintech, Rosemont, IL, United States, 17507-1-AP), anti-Tubulin (Abcam, 5335s), and horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Danvers, MA, United States).

Techniques: Expressing, Over Expression

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Detailed view of blocking loop 2 (BL2), which occludes the active site of USP14 (PDB access code 2AYN). The BL2 loop, which contains Ser432, is shown in stick model, in the apo form. ( B ) Combined ribbon representation and stick model showing a comparison of the conformations of the BL2 loop contained in the apo form (blue, PDB access code 2AYN) and in the USP14- Ub-aldehyde (Ubal) adduct (orange, PDB access code 2AYO). In this drawing, the Ser432 and Cys114 residues are shown in stick model, and the bound Ubal (a ubiquitin derivative in which the C-terminal carboxylate is replaced by an aldehyde) in the complex is drawn in green. ( C ) A surface charge potential representation (contoured at ± 7 kT/eV; blue/red) of USP14 (PDB accession 2AYN) showing that the S432 residue is very close to a highly negatively charged patch mainly formed by the acidic E188, D199, and E202 residues. When S432 is phosphorylated, the negatively charged phosphate group may induce a repulsive force, thereby relieving inhibition of the catalytic activity of USP14. ( D ) USP14 domain organization and sequence alignment of the Akt phosphorylation site within USP14 orthologs from different species. Two BLs (BL1 and BL2) covering the USP14 active site are shown. The Akt phosphorylation site in USP14 from different species as predicted by Scansite. ( E ) S432 is the major phosphorylation site in USP14. HEK293T cells were treated as in , followed by electrospray ionization mass spectrometry (ESI-MS) analysis. Spectral counts were determined by ESI-MS. ( F ) Akt phosphorylates USP14 in vitro. Bacterially expressed and purified USP14 was incubated with active Akt in the presence of ATP. Reaction products were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and phosphorylated species were detected by a phospho-Ser antibody. DOI: http://dx.doi.org/10.7554/eLife.10510.003

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Blocking Assay, Comparison, Ubiquitin Proteomics, Residue, Inhibition, Activity Assay, Sequencing, Phospho-proteomics, Mass Spectrometry, In Vitro, Purification, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Akt interacts with USP14. HEK293T cells were transfected with indicated plasmids for 24 hr. The cell lysates were collected for co-immunoprecipitation and western blotting analysis. ( B ) Schematic representation of mass spectrometry assay to determine USP14 phosphorylation sites by Akt. ( C ) Four phosphorylation sites of USP14 were determined by mass spectrometry. ( D ) The representative MS/MS spectrum of phosphorylated tryptic peptide ‘SSSphosSGHYVSWVK’ of human USP14 protein. The peptide sequence ‘SSSphosSGHYVSWVK’ containing phosphorylated S432 was identified by shotgun analysis using mass spectrometry when USP14 was coexpressed with Myr-Akt in HEK293T cells. Fragmentation ion of the amide bond of the peptide result in formation of “b” ion and “y” ion series corresponding to the N- and C-terminal fragments respectively. Representative ions with phosphorylation and H 2 O loss were manually labeled in red on the spectrum. DOI: http://dx.doi.org/10.7554/eLife.10510.004

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Transfection, Immunoprecipitation, Western Blot, Mass Spectrometry, Phospho-proteomics, Tandem Mass Spectroscopy, Sequencing, Labeling

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) In vitro phosphorylation of USP14 at S432 by Akt. Bacterially expressed and purified wild type USP14 or AA mutant incubated with active Akt in the presence of ATP. Reaction products were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and phosphorylation was detected by the phospho-Ser antibody. ( B ) Akt phosphorylates USP14 at S432 in vivo. Western blot analysis of whole cell lysate and immunoprecipitates derived from HEK293T cells transfected with wild type USP14, USP14 S143A, USP14 S432A, and USP14 S143A/S432A (AA) constructs using the phospho-Ser antibody. L.E., long exposure. ( C ) Immunoprecipitation (IP) and IB analysis of HEK293T cells transfected with HA-USP14 and Myr-Akt and preincubated with or without λ-phosphatase as indicated. ( D ) Inhibition of Akt decreased exogenous USP14 phosphorylation. HEK293T cells were transfected with Myc-USP14 for 20 hr then treated with 1 μM MK2206 or deprived of serum for another 4 hr before harvest. ( E ) In vitro kinase assay to detect Akt phosphorylation of USP14 by phospho-Ser432-specific antibody and phos-tag-containing gels. Bacterially expressed and purified wild type USP14 or S432A mutant was incubated with active Akt in the presence of ATP. The reaction products were resolved by SDS-PAGE, and USP14 phosphorylation was detected using an antibody that specifically recognizes Ser432 phosphorylation of USP14 or determined by differential migration on phos-tag gels. ( F ) In vivo detection of endogenous USP14 Ser432 phosphorylation by anti-p-Ser432-specific antibody. Western blot analysis of immunoprecipitates derived from H4 cells transfected with or without Myr-Akt plasmids using the anti-p-Ser432-specific antibody. ( G, H ) Phosphorylation of endogenous USP14 S432 upon stimulation with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF). HEK293T cells were serum-starved and pretreated with Akt inhibitor MK2206 (1 μM) for 30 min before stimulation with IGF-1 (100 ng/mL) for 30 min ( G ) or EGF (100 ng/mL) for 1 hr ( H ). The cell lysates were immunoprecipitated with USP14 antibody and western-blotted with anti-p-S432 antibody. ( I ) Phosphorylation of endogenous USP14 S432 in Pten knockout cells with high activity of Akt. Lysates from mouse embryonic fibroblasts (MEFs) with indicated genotypes were immunoprecipitated with USP14 antibody and then Western blotted with p-S432 antibody. The differential migration of phospho-USP14 on phos-tag-containing gels was determined as shown in the bottom panel. DOI: http://dx.doi.org/10.7554/eLife.10510.005

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: In Vitro, Phospho-proteomics, Purification, Mutagenesis, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, In Vivo, Western Blot, Derivative Assay, Transfection, Construct, Immunoprecipitation, Inhibition, Kinase Assay, Migration, Knock-Out, Activity Assay

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Akt phosphorylates USP14 at S432 in vivo. Western blotting analysis of whole cell lysate and immunoprecipitates derived from HEK293T cells transfected with wild type USP14, USP14 S143A, USP14 S432A, and USP14 S143A/S432A (AA) constructs using an Akt phosphorylation-consensus motif (R××S/T) antibody. (B, C) Inhibition of Akt decreases USP14 S432 phosphorylation levels. H4 cells were treated with different concentration of Akt inhibitors MK2206 ( B ) or AZD5363 ( C ) as indicated for 4 hr. The cell lysates were collected for immunoprecipitation and western blotting analysis. ( D ) Inhibition of phosphoinositide 3-kinases (PI3K) decreases USP14 S432 phosphorylation levels. H4 cells were treated with different concentration of PI3K inhibitors GDC0941 or Wortmannin as indicated for 4 hr. The cell lysates were collected for immunoprecipitation and western blotting analysis. ( E ) ERK1/2 inhibition has no effect on USP14 S432 phosphorylation. H4 cells were treated with different concentration of ERK1/2 inhibitor U0126 as indicated for 4 hr. The cell lysates were collected for immunoprecipitation and western blotting analysis. ( F ) Pten –/– mouse embryonic fibroblast (MEF) cells were treated with 1 μM Akt inhibitors for 4 hr, then cell lysates were collected for immunoprecipitation and western blotting analysis. DOI: http://dx.doi.org/10.7554/eLife.10510.006

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: In Vivo, Western Blot, Derivative Assay, Transfection, Construct, Phospho-proteomics, Inhibition, Concentration Assay, Immunoprecipitation

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A, B ) A representative curve of USP14 recombinant protein purification before ( A ) and after ( B ) cleavage by 3C protease on size-exclusion chromatography. Bacterially expressed USP14 forms oligomer and dimer, only monomer was used for further experiments. Trx-tag was removed to get tag-free USP14 protein for in vitro kinase assay or Ub-AMC and ubiquitin cleavage assay. ( C ) One dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining of purified USP14 protein before and after cleavage by 3C protease. DOI: http://dx.doi.org/10.7554/eLife.10510.008

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Recombinant, Protein Purification, Size-exclusion Chromatography, In Vitro, Kinase Assay, Ubiquitin Proteomics, Cleavage Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Purification

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Akt activates USP14 DUB activity in vitro. USP14 protein (1 μg) was incubated with or without active Akt (1 μg) in kinase assay buffer in a total volume of 50 μL for 1 hr at 30 ° C, then the reaction mixtures were subjected to Ub-AMC assay. RFU, relative fluorescence units. (B, C) Akt activates USP14 in cells. USP14 was immunoprecipitated from HEK293T cells coexpressed with activated Akt ( B ) or treated with 10 μM MK2206 for 4 hr ( C ) and then eluted with HA-peptide following Ub-AMC hydrolysis assay. ( D ) Activation of USP14 by stimulating cells with IGF-1. HEK293T cells were serum-starved and pretreated with or without Akt inhibitor MK2206 (1 μM) for 30 min before stimulation with IGF-1 (100 ng/mL) for 30 min. USP14 was then immunoprecipitated and eluted with HA-peptide. The activity of USP14 was determined using Ub-AMC hydrolysis assay. ( E ) USP14 activation by Akt is blocked by S432A mutation. Ub-AMC hydrolysis assay of wildde type USP14 or S432A mutant in the presence or absence of active Akt. ( F ) Ub-AMC hydrolysis assay of bacterially expressed and purified wild type USP14 or S432E mutant. ( G ) Lineweaver–Burk analysis of USP14 S432E, obtained by measuring the initial rates at varying Ub-AMC concentrations (see for reference). ( H ) The activity of phospho-mimetic USP14 mutant can be further stimulated by the presence of proteasome. Ub-AMC hydrolysis assay of wild type USP14 or S432E mutant in the presence or absence of Ub-VS-treated human proteasome (VS-proteasome (see ); 1 nM). Ptsm, 26S proteasome. DOI: http://dx.doi.org/10.7554/eLife.10510.007

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Activity Assay, In Vitro, Incubation, Kinase Assay, Ub-AMC Assay, Fluorescence, Immunoprecipitation, Hydrolysis Assay, Activation Assay, Mutagenesis, Purification

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Validation of Ub-AMC assay on immunoprecipitated USP14 from HEK293T cells. Wild type or inactive mutant USP14 was immunoprecipitated from transfected HEK293T cells and then eluted with HA-peptide following Ub-AMC hydrolysis assay. ( B–D ) Ser143 phosphorylation has limited effect on USP14 activity. S143A mutant had similar levels of hydrolyzing activity as that of WT USP14 in the presence of active Akt ( B ). Double E mutant (S143E/S432E) showed similar levels of hydrolyzing activity as that of S432E single mutant ( C ). S143E mutant has minor effect on USP14 activity compared with S432E mutant ( D ). ( E ) Linear kinetics (R 2 > 0.99) of Ub-AMC hydrolysis. ( F ) Distribution of total and phosphorylated USP14 specific in glycerol gradient centrifugation. Soluble lysates of Pten –/– mouse embryonic fibroblast (MEF) cells, prepared as described in Materials and methods, were subjected to glycerol density gradient centrifugation. Gradient fractions were collected and subjected to western blotting with the indicated antibodies. Anti-RPN11 was used as a control for proteasome. DOI: http://dx.doi.org/10.7554/eLife.10510.009

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Biomarker Discovery, Ub-AMC Assay, Immunoprecipitation, Mutagenesis, Transfection, Hydrolysis Assay, Phospho-proteomics, Activity Assay, Gradient Centrifugation, Western Blot, Control

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Dimeric Ub cleavage assay. USP14 S432E cleavage of Lys 48 and Lys 63 Ub chain linkages was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). ( B ) Cleavage of Lys 48, Lys 63 and linear dimeric Ub chain types in the presence of USP14 S432E was measured over time and analyzed using SDS-PAGE. Quantification of the amount of dimer remaining from the data was shown below. ( C ) Dimeric Ub cleavage analysis of immunoprecipitates derived from HEK293T cells transfected with wild type HA-USP14 or S432A mutant plasmids. DOI: http://dx.doi.org/10.7554/eLife.10510.010

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Cleavage Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Derivative Assay, Transfection, Mutagenesis

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) USP14 S432E does not cleave linear di-Ub. USP14 S432E cleavage of linear dimeric Ub chain types and analyzed using SDS-PAGE. Cleavage of Lys48 dimeric Ub chain types was used as a positive control. ( B, C ) USP14 immunoprecipitated from transfected HEK293T cells and eluted with HA-peptide has high deubiquitination activity towards both K48 and K63 di-Ub but not linear di-Ub. DOI: http://dx.doi.org/10.7554/eLife.10510.011

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: SDS Page, Positive Control, Immunoprecipitation, Transfection, Activity Assay

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: ( A ) Inhibition of Akt promotes UPS function. H4-GFP-CL1 cells were treated with different concentrations of MK2206 as indicated for 4 hr. The cells were then harvested and subjected to western blotting analysis using indicated antibodies. ( B ) H4-GFP-CL1 cells were treated as in ( A ) and . Images of the cells were collected using an ArrayScan HCS 4.0 Reader. The average GFP intensity in 2,000 cells from each indicated sample was determined. Data are displayed as mean ± SD of the GFP intensity per cell. ** p <0.01, *** p <0.001 ( C ) Inhibition of Akt or phosphoinositide 3-kinase (PI3K) promotes UPS function. H4-GFP-CL1 cells were treated with Akt inhibitor AZD5363 (1 μM) or PI3K inhibitor GDC0941 (1 μM) as indicated for 4 hr. The cells were then harvested and subjected to western blotting analysis using indicated antibodies. p-GSK3β(S9) was blotted to indicate the inhibition of Akt. ( D ) Activation of Akt inhibits UPS. H4-GFP-CL1 cells were transfected with Myr-Akt for 24 hr. The cells were then harvested and subjected to western blotting analysis using indicated antibodies. ( E ) IGF-1 stimulation inhibits UPS function. H4-GFP-CL1 cells were serum-starved and pretreated with Akt inhibitor MK2206 (1 μM) for 30 min before stimulating with IGF-1 (100 ng/mL) for 30 min. The cells were then imaged and quantified as in (B). ( F ) H4-GFP-CL1 cells were treated as in (D) and . Then cells were imaged and quantified as in (B). ( G ) EGF stimulation inhibits UPS function. H4-GFP-CL1 cells were serum-starved and pretreated with Akt inhibitor MK2206 (1 μM) for 30 min before stimulation with EGF (100 ng/mL) for 1 h. The cells were then harvested and subjected to western blotting analysis using indicated antibodies. ( H ) Akt regulates UPS function through USP14. Myr-Akt was transfected into either wild type or Usp14 –/– H4 cells stably expressing GFP-CL1 for 24 hr. The cells were then harvested and subjected to western blotting analysis using indicated antibodies. ( I ) Akt regulates UPS function through phosphorylation of USP14. Myr-Akt was transfected into either wild type USP14 or USP14 AA reconstitution cell lines stably expressing GFP-CL1 for 24 hr. The cells were then imaged and quantified as in (B). DOI: http://dx.doi.org/10.7554/eLife.10510.012

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Inhibition, Western Blot, Activation Assay, Transfection, Stable Transfection, Expressing, Phospho-proteomics

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: (A, B, C, D) Generation of USP14 knockout H4 cell line using CRISPR/Cas9 system. A schematic of the Cas9/sgRNA/oligo targeting site in the exon2 of Usp14 . The sgRNA coding sequence is underlined and labeled in red. The protospacer-adjacent motif (PAM) sequence is underlined ( A ). The deleted sequences in the Usp14 –/– cell lines are presented. The number of sequences analyzed is indicated right ( B ). Sequencing analysis of Usp14 –/– cell lines. The arrow indicates the missing sequences (TCAAG) ( C ). Western blotting analysis of USP14 expression in WT cells and Usp14 –/– cells ( D ). ( E ) Akt regulates UPS function through phosphorylation of USP14. An expression vector for Myr-Akt was transfected into either wild type USP14 or USP14 AA reconstitution cell lines stably expressing GFP-CL1 and incubated for 24 hr. Then the cell lysates were harvested and subjected to western blotting analysis using indicated antibodies. ( F ) GFP-cODC assay was verified. H4-GFP-cODC cells were treated with 10 μM MG132 for 4 hr, and then the cell lysates were harvested and subjected to western blotting analysis using indicated antibodies. ( G ) H4-GFP-cODC cells were treated with 10 μM MK2206 for 4 hr or transfected with an expression vector for Myr-Akt as indicated for 24 hr. Then the cell lysates were harvested and subjected to western blotting analysis using indicated antibodies. DOI: http://dx.doi.org/10.7554/eLife.10510.014

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Knock-Out, CRISPR, Sequencing, Labeling, Western Blot, Expressing, Phospho-proteomics, Plasmid Preparation, Transfection, Stable Transfection, Incubation

Journal: eLife

Article Title: Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

doi: 10.7554/eLife.10510

Figure Lengend Snippet: The quantitative analysis of proteome change in USP14 knockout or USP14 mutant cells were performed by tandem mass tag (TMT)-isobaric labeling followed by shotgun analysis. The heat map was plotted based on the set of 87 proteins that are down-regulated greater than or equal to 1.2-fold in H4 KO cells compared to H4 WT cells or to H4 KO cells complemented with WT USP14 (KO-WT). The log base 2 of average ratios was plotted as indicated. DOI: http://dx.doi.org/10.7554/eLife.10510.015

Article Snippet: Phospho-USP14 S432 antibodies were generated by Proteintech.

Techniques: Knock-Out, Mutagenesis, Labeling

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: ST14 expression in different datasets from patients with ovarian cancer. ( A ) Oncomine analysis of the mRNA expression levels of ST14 genes in different cancers. The differences in expression levels of genes between cancer and normal tissues are concluded. The thresholds are indicated in the colored cells. P < 0.05, fold-change > 2 and gene rank = 10% were considered statistically significant. Red cells represent overexpression of the target gene in tumor tissues compared to normal tissues, while blue cells indicate downregulation of the gene. Gene rank is depicted by the color depth in the cells. ( B ) UALCAN analysis of the mRNA expression levels of ST14 genes in different cancers. ( C ) GEPIA analysis of the mRNA expression levels of ST14 genes in different cancers. ( D ) ST14 DNA copy numbers based on chips for ovarian cancer research in TCGA Ovary. * P < 0.05. ( E ) Levels of ST14 mRNA in ovarian cancer based on research in the GEPIA websites (red for tumor, black for normal). The boxplot analysis showed the expression level by log2 (TPM + 1) on a log-scale. * P < 0.05. ( F ) Correlation between ST14 and TMEFF1 expression in ovarian cancer based on the GEPIA website. R = 0.32, *** P < 0.001. ( G ) The expression of ST14 and TMEFF1 in ovarian malignant tumor tissues and normal tissues detected by western blot. ( H ) Quantification of TMEFF1 normalized to GAPDH. Data are presented as the mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques: Expressing, Over Expression, Western Blot

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Levels of ST14 in subgroups of patients with ovarian cancer. Levels of ST14 expression in ovarian cancer patients based on different ( A ) ages, ( B ) races, ( C ) tumor grades, ( D ) cancer stages, and ( E ) TP53 methylation statuses. OV, ovarian serous cystadenocarcinoma. * P < 0.05

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques: Expressing, Methylation

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Differentially expressed genes correlated with ST14 in ovarian cancer. ( A ) Correlations between ST14 and genes differentially expressed in ovarian cancer were assessed by the Pearson test. ( B ) Genes positively correlated with ST14 in ovarian cancer as heat maps (TOP 50). Red: positively correlated genes. Blue: negatively correlated genes. ( C ) Genes negatively correlated with ST14 in ovarian cancer as heat maps (TOP 50). ( D - F ) Correlation between ST14 expression and the expression of EI24 ( D ), SRPR ( E ), and ESRP1 ( F ) based on the Pearson test, shown with a scatter plot ( P = 6.457e-29, P = 3.555e-25, P = 6.453e-26, respectively)

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques: Expressing

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Significantly enriched GO annotations and KEGG pathways of ST14-co-expressed genes and proteins interacting with ST14 in ovarian cancer. Results were analyzed with Metascape. The top 20 enriched ( A ) cellular components, ( C ) biological processes, and ( E ) molecular functions related to ST14-related genes are shown, with the bar graph colored based on P -values. ( B , D , F ) Network of GO-enriched terms colored based on the P -value, where terms containing more genes tended to have a more significant P -value. ( G ) KEGG-enriched terms colored based on P -values. ( H ) Network of KEGG-enriched terms colored based on the P -value, where terms containing more genes tended to have a more significant P -value. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes. ( I ) PPI analysis using the STRING database. ( J ) PPI analysis using the GeneMANIA database

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques:

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Analysis of ST14 genetic variations and effect on survival and prognosis of ovarian cancer patients. ( A ) Mutations in the ST14 gene based on the cBioPortal database. ( B ) Analyses of genetic variations in ST14 reported in different studies. The variations included mutation (green), amplification (red), and deep deletions (blue). TCGA: The Cancer Genome Atlas. ( C - F ) Effect of mutations in the ST14 gene on the ( C ) overall survival (OS), ( D ) disease-free survival (DFS), ( E ) disease-specific survival (DSS), and ( F ) progression-free survival (PFS) of ovarian cancer patients ( P > 0.05)

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques: Mutagenesis, Amplification

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Expression and co-localization of ST14 and TMEFF1 in different ovarian tissues. ( A ) Immunohistochemical staining of ovarian malignant tumors (i, v), borderline tumors (ii, vi), benign tumors (iii, vii), and normal ovarian tissues (vi, viii). ST14 (i-iv) and TMEFF1 (v-viii) staining is shown (original magnification, ×400). ( B ) Dual-labeled immunofluorescence technology was used to detect the co-localization of ST14 and TMEFF1 in different ovarian tissues. Blue represents the nucleus, red represents ST14, green represents TMEFF1, orange represents the co-localization of ST14 and TMEFF1 (original magnification, ×400). Pearson’s correlation coefficient (Rr) and Manders’ overlap coefficient (R) of the co-localization images: malignant tumors (Rr:0.64, R:0.87), borderline tumors (Rr:0.76,R:0.79), benign tumors (Rr:0.63,R:0.68), and normal ovarian tissues (Rr:0.76,R:0.92)

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques: Expressing, Immunohistochemical staining, Staining, Labeling, Immunofluorescence

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Expression of ST14 in different ovarian tissues

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques: Expressing

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Association between ST14 expression and pathological features in ovarian cancer

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques: Expressing

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Univariate Kaplan-Meier prognostic analysis of ovarian cancer

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques: Biomarker Discovery

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of patients with ovarian cancer

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques: Biomarker Discovery

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Relevance of ST14 and TMEFF1 expression in ovarian cancer

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques: Expressing

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Co-localization and interaction of ST14 and TMEFF1 in ovarian cancer cells, and ST14 regulates the expression of TMEFF1. ( A ) Dual-labeled immunofluorescence technology was used to detect the co-localization of ST14 and TMEFF1 in ovarian cancer cell. Blue represents the nucleus, red represents ST14, green represents TMEFF1, and orange represents the co-localization of ST14 and TMEFF1 (original magnification, ×600). Pearson’s correlation coefficient (Rr) and Manders’ overlap coefficient (R) of the co-localization images are Rr:0.73, R:0.61. (B-C) The cell lysates of CAOV3, OVCAR3, and SKOV3 cells were immunoprecipitated with an anti-TMEFF1 antibody ( B ) and anti-ST14 antibody ( C ), and then, western blotting was performed with an anti-ST14 antibody and anti-TMEFF1 antibody. “Input” is the total cell lysate of CAOV3 cells. “IgG” is the negative control. ( D ) In the ovarian cancer cell lines CAOV3 and SKOV3, the expression of TMEFF1 decreased after knocking down the ST14 gene. ( E ) Quantification of ST14 and TMEFF1 normalized to GAPDH. Representative images and accompanying statistical plots are presented. Blank, blank control group, untreated original cells; siST14, ST14 gene knockdown group (through siRNA); NC, negative control group, negative gene (no sequence homology with ST14) knockdown group (through siRNA). Data are presented as the mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques: Expressing, Labeling, Immunofluorescence, Immunoprecipitation, Western Blot, Negative Control, Control, Knockdown, Sequencing

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: Interaction between ST14 and TMEFF1 promotes proliferation, invasion and migration of ovarian cancer. ( A , B , E , F ) The invasion capacities of ovarian cancer cells (CAOV3 and SKOV3) after downregulation of ST14 protein and addition of recombinant TMEFF1 active protein detected by Transwell assay. Number 1,2,3,4 respectively represents CAOV3-NC, CAOV3-siST14, CAOV3-NC + TMEFF1 and CAOV3-siST14 + TMEFF1; Number 5,6,7,8 respectively represents SKOV3-NC, SKOV3-siST14, SKOV3-NC + TMEFF1 and SKOV3-siST14 + TMEFF1. ( C , D , G , H ) The migration capacities of ovarian cancer cells (CAOV3 and SKOV3) after downregulation of ST14 protein and addition of recombinant TMEFF1 active protein detected by Wound healing assay. Number 1,2,3,4 respectively represents CAOV3-NC, CAOV3-siST14, CAOV3-NC + TMEFF1 and CAOV3-siST14 + TMEFF1; Number 5,6,7,8 respectively represents SKOV3-NC, SKOV3-siST14, SKOV3-NC + TMEFF1 and SKOV3-siST14 + TMEFF1. ( I - J ) The proliferation capacities of ovarian cancer cells (CAOV3 and SKOV3) after downregulation of ST14 protein and addition of recombinant TMEFF1 active protein detected by MTT assay. Data are presented as the mean ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques: Migration, Recombinant, Transwell Assay, Wound Healing Assay, MTT Assay

Journal: BMC Cancer

Article Title: ST14 interacts with TMEFF1 and is a predictor of poor prognosis in ovarian cancer

doi: 10.1186/s12885-024-11958-8

Figure Lengend Snippet: The kinase, miRNA, and transcription factor-target networks of ST14 in ovarian cancer

Article Snippet: After blocking with 5% milk for 1 h, the PVDF membrane was incubated with the primary antibody at 4 °C for 14 h. The primary antibodies were as follows: anti-TMEFF1 antibody (Santa Cruz, 1:500, catalog number 393,457), anti-ST14 antibody (Proteintech, rabbit, catalog number 27176-1-AP), anti-GAPDH (ZSBIO, China, 1:2000, catalog number TA08).

Techniques: Phospho-proteomics