primary antibodies against timp2  (R&D Systems)

Journal: Biology of Reproduction

Article Title: Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC 1

doi: 10.1095/biolreprod.111.093484

Figure Lengend Snippet: TABLE 1.

Article Snippet: Total cellular proteins and nuclear extracts were examined using primary antibodies against TIMP2 (1:500 dilution; product no. AF971; R&D Systems, Inc., Cambridge, MA), FSH receptor (FSHR; 1:1000 dilution; product no. ab65975; Abcam, Inc., Cambridge, MA), CEBPA (1:1000 dilution; product no. 2295; Cell Signaling Technology, Inc., Beverly, MA), cAMP response element-binding protein (CREB; 1:500 dilution; product no. 9197; Cell Signaling Technology, Inc.), MYC (c-Myc; 1:1000 dilution; product no. 5605; Cell Signaling Technology, Inc.), TATA box binding protein (TBP; 1:1000 dilution; product no. ab63766; Abcam, Inc.), and α-tubulin (1:2000 dilution; product no. 2144; Cell Signaling Technology) coupled with horseradish peroxidase-conjugated secondary antibody (1:5000 dilution; Santa Cruz Biotechnology, Inc.).

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Corylin Attenuates CCl 4 -Induced Liver Fibrosis in Mice by Regulating the GAS6/AXL Signaling Pathway in Hepatic Stellate Cells

doi: 10.3390/ijms242316936

Figure Lengend Snippet: Corylin inhibits HSC activation by suppressing GAS6 expression and downstream PI3K/AKT signaling. ( A ) Western blotting results of cell lysates of HHSteC cells treated with corylin or DMSO for 2 h, followed by TGF-β (4 ng/mL) treatment for 24 h to stimulate cell activation and to determine the effects of corylin on the GAS6/AXL signaling pathway. β-Actin is the internal control. Quantitative results are shown in ( B , C ). All data are expressed as the mean ± standard deviations of three independent experiments. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). HSCs, hepatic stellate cells; GAS6, growth arrest-specific gene 6; PI3K/AKT, phosphoinositide 3-kinase/protein kinase B; p-PI3K, phosphorylated PI3K; DMSO, dimethyl sulfoxide; TGF-β, transforming growth factor beta; COL1A1, collagen 1A; α-SMA, smooth muscle-actin.

Article Snippet: Primary antibodies against AXL, phosphorylated (phospho)-AXL, GAS6, phospho-PI3K, PI3K, phospho-AKT, AKT, IL-1β, IL-6, MMP-2, MMP-9, TIMP-1, TIMP2, cleaved caspase-3, caspase-3, cleaved caspase-9, and caspase-9 were purchased from Genetex (Irvine, CA, USA), ABclonal (Woburn, MA, USA), and Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Control

Journal: Molecular Therapy Oncolytics

Article Title: Inhibition of tumor invasion and metastasis by targeting TGF-β-Smad-MMP2 pathway with Asiatic acid and Naringenin

doi: 10.1016/j.omto.2021.01.006

Figure Lengend Snippet: Combination of AA and NG further reduces MMPs expressions in LLC tumor compared with individual therapies (A) Western blot detecting protein levels of p-Smad3, Smad7, MMP2, MMP9, and MMP13 in LLC tumor. (B–E) mRNA levels of (B) MMP2, (C) MMP3, (D) MMP9, and (E) MMP13 in LLC tumor. Each bar represents the mean ± SD for groups of three to four mice. ∗p < 0.05, ∗∗p < 0.01 compared to Ctrl; #p < 0.05 as indicated.

Article Snippet: After blocking with 5% BSA/Tris-buffered saline (TBS) buffer, membranes were incubated with primary antibodies against mouse p-Smad3, Smad7, p-ALK5 (Abcam, MA, USA), MMP2, MT1-MMP, TIMP2 (Merck Millipore, MA, USA), MMP9, MMP13, β-actin (Santa Cruz Biotechnology, CA, USA), and NF-κB p50 (Cell Signaling, MA, USA) dissolved in 1% BSA overnight at 4°C, followed by incubating with IRDye 800-conjugated secondary antibody (Rockland Immunochemicals, PA, USA).

Techniques: Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Inhibition of tumor invasion and metastasis by targeting TGF-β-Smad-MMP2 pathway with Asiatic acid and Naringenin

doi: 10.1016/j.omto.2021.01.006

Figure Lengend Snippet: Rebalancing Smad3/Smad7 signaling with AA and NG produces better inhibitory effects on TGF-β1-induced MMP2 transcription, activation, and function (A and B) Western blot detecting expression of (A) p-ALK5, p-Smad3, and pro- and active-MMP2 with different doses of TGF-β1 stimulation, (B) p-ALK5, p-Smad3, Smad7, NF-κB p50, pro- and active-MMP2 (M2), MT1-MMP, and TIMP2 in LLC cells with AA and NG treatment under TGF-β1 stimulation at 5 ng/mL. (C and D) Real-time PCR shows TGF-β1 induces MMP2 transcription in a dose-dependent manner, which is inhibited by AA, NG, and combined therapy (CB). Each bar represents the mean ± SD for groups of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to TGF-β1; #p < 0.05, ##p < 0.01, ###p < 0.001 as indicated.

Article Snippet: After blocking with 5% BSA/Tris-buffered saline (TBS) buffer, membranes were incubated with primary antibodies against mouse p-Smad3, Smad7, p-ALK5 (Abcam, MA, USA), MMP2, MT1-MMP, TIMP2 (Merck Millipore, MA, USA), MMP9, MMP13, β-actin (Santa Cruz Biotechnology, CA, USA), and NF-κB p50 (Cell Signaling, MA, USA) dissolved in 1% BSA overnight at 4°C, followed by incubating with IRDye 800-conjugated secondary antibody (Rockland Immunochemicals, PA, USA).

Techniques: Activation Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Respiratory Research

Article Title: Influence of obesity on remodeling of lung tissue and organization of extracellular matrix after blunt thorax trauma

doi: 10.1186/s12931-020-01502-0

Figure Lengend Snippet: Gene expression status of Mmps and Timps in lung tissue post-BTT. Expression levels of Mmp2 , Mmp8 , Mmp9 , Timp2 and Timp1 in lung tissue were determined by qPCR using Gapdh as a housekeeping gene. Mmp2 , Mmp8 , Mmp9 , Timp1 and Timp2 show the gene expression differences by comparing all the time-points after lung injury to their respective controls. Statistical significance was determined using two-way ANOVA followed by an uncorrected Fisher’s LSD test (α = 0.05). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. Indicators of significance directly above the bars of the diagram indicate a statistical difference to the control, while stars above the connector line show differences between the two diets at that specific time point

Article Snippet: Sections were washed in PBS and subsequently stained overnight at 4 °C with primary antibodies either against TIMP1 (ITT4658, G-Biosciences, 1:500) or TIMP2 (ITA6535, G-Biosciences, 1:200).

Techniques: Gene Expression, Expressing, Control

Journal: Respiratory Research

Article Title: Influence of obesity on remodeling of lung tissue and organization of extracellular matrix after blunt thorax trauma

doi: 10.1186/s12931-020-01502-0

Figure Lengend Snippet: TIMP1/2 IHC staining of lung from lean and obese mice after BTT. A) TIMP1 and B) TIMP2 staining in lean and obese mice from control animals as well as 1 h, 6 h, 24 h, 72 h and 192 h post trauma. The area of positive staining was measured and is depicted as values next to the respective depiction of the staining for TIMP1 (C) and TIMP2 (D). The data is presented as mean ± SEM. Statistical significance was determined using two-way ANOVA followed by an uncorrected Fisher’s LSD test (α = 0.05) ** indicates p < 0.01

Article Snippet: Sections were washed in PBS and subsequently stained overnight at 4 °C with primary antibodies either against TIMP1 (ITT4658, G-Biosciences, 1:500) or TIMP2 (ITA6535, G-Biosciences, 1:200).

Techniques: Immunohistochemistry, Staining, Control

Journal: Scientific Reports

Article Title: EZH2-mediated epigenetic silencing of TIMP2 promotes ovarian cancer migration and invasion

doi: 10.1038/s41598-017-03362-z

Figure Lengend Snippet: EZH2 inversely correlates with TIMP2 and indicates poor prognosis. ( a ) Representative images of immunohistochemistry analysis of EZH2 and TIMP2 in ovarian cancer tissues. EZH2 was expressed in the nucleus, while TIMP2 was expressed in the cytoplasm. Sample No. 174 has high EZH2 expression and low TIMP2 expression. Sample No. 338 has low EZH2 expression and high TIMP2 expression (scale bar, 50 μm). ( b ) Negative correlation between the percentage of EZH2-positive cells and the TIMP2 staining score in ovarian cancer tissues (Spearman’s correlation test). ( c – e ) Kaplan-Meier plots for overall survival in epithelial ovarian cancer patients. High EZH2 ( c ), low TIMP2 ( d ) or high EZH2 and simultaneous low TIMP2 ( e ) are associated with reduced overall survival. ( f – h ) Kaplan-Meier plots for progression-free survival in patients with epithelial ovarian cancer stratified by the expression status of EZH2 ( f ), TIMP2 ( g ), or both EZH2 and TIMP2 ( h ).

Article Snippet: Then, the slides were incubated with primary antibody against EZH2 (1:200 dilution, Cell Signaling Technology, Danvers, MA, USA) and TIMP2 (1:200 dilution, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Immunohistochemistry, Expressing, Staining

Journal: Scientific Reports

Article Title: EZH2-mediated epigenetic silencing of TIMP2 promotes ovarian cancer migration and invasion

doi: 10.1038/s41598-017-03362-z

Figure Lengend Snippet: EZH2 regulates TIMP2 expression in vitro . ( a ) Western blot analysis of EZH2, TIMP2, MMP2, and MMP9 protein expression in ovarian cancer cell lines. Histone-3 (H3) and β-actin serve as internal controls. The cropped blots are used in the figure, and full-length blots are presented in Supplementary Fig. . ( b ) EZH2 and TIMP2 mRNA expression levels in seven ovarian cancer cell lines were detected by quantitative real-time PCR. ( c ) Negative correlation between TIMP2 and EZH2 mRNA levels in ovarian cancer cell lines. ( d ) qRT-PCR for EZH2 and TIMP2 expression in A2780 and SKOV3 cells with EZH2 up- or down-regulation by transfection with an EZH2-overexpressing plasmid (EZH2) or targeting shRNA (shEZH2). ( e ) Western blot analysis of EZH2, TIMP2 and MMP2/9 protein expression in EZH2-regulated A2780 and SKOV3 cells. Histone-3 (H3) and β-actin are used as internal controls. The cropped blots are used in the figure, and full-length blots are presented in Supplementary Fig. . ( f ) Immunocytochemical staining of EZH2 and TIMP2 in EZH2-regulated A2780 and SKOV3 cells (scale bar, 20 μm). ( g ) Gelatin zymography assays of MMP2 and MMP9 activity in EZH2-regulated A2780 and SKOV3 cells. The cropped gels are used in the figure, and full-length gels are presented in Supplementary Fig. . * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Then, the slides were incubated with primary antibody against EZH2 (1:200 dilution, Cell Signaling Technology, Danvers, MA, USA) and TIMP2 (1:200 dilution, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Expressing, In Vitro, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Plasmid Preparation, shRNA, Staining, Zymography, Activity Assay

Journal: Scientific Reports

Article Title: EZH2-mediated epigenetic silencing of TIMP2 promotes ovarian cancer migration and invasion

doi: 10.1038/s41598-017-03362-z

Figure Lengend Snippet: EZH2 promotes ovarian cancer cell invasion and migration by repressing TIMP2. Cells were transfected with shEZH2, shEZH2 and siTIMP2; EZH2-plasmid, EZH2-plasmid and TIMP2-plasmid and corresponding controls. ( a ) Western blot analysis of EZH2 and TIMP2 in SKOV3 cells after co-transfection. The cropped blots are used in the figure, and full-length blots are presented in Supplementary Fig. . ( b ) Scratch-wound assays for the detection of cell mobility (scale bar, 100 μm). ( c ) Histograms show the wound-healing areas of the scratch-wound assays. ( d ) Transwell migration and invasion assays. The number of invading and migrating cells is presented. ( e ) Representative images of migration and invasion assays (scale bar, 50 μm). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Then, the slides were incubated with primary antibody against EZH2 (1:200 dilution, Cell Signaling Technology, Danvers, MA, USA) and TIMP2 (1:200 dilution, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Migration, Transfection, Plasmid Preparation, Western Blot, Cotransfection

Journal: Scientific Reports

Article Title: EZH2-mediated epigenetic silencing of TIMP2 promotes ovarian cancer migration and invasion

doi: 10.1038/s41598-017-03362-z

Figure Lengend Snippet: Down-regulation of EZH2 inhibits ovarian cancer metastasis by elevating TIMP2 in xenograft models. Nude mice were intraperitoneally inoculated with SKOV3 cells stably transfected with shNC or shEZH2. In vitro transient transfection and in vivo transfection of siTIMP2 was performed in one subgroup inoculated with SKOV3-shEZH2. ( a ) Peritoneal dissemination of xenografts. SKOV3-shEZH2 cells formed many fewer i.p. metastatic nodules (blue arrow) in the abdominal cavity of mice than SKOV3-shNC cells. In contrast, the transfection of siTIMP2 could significantly increase the reduced tumor metastatic nodules mediated by EZH2 depletion. ( b ) Representative images of the mesenteric membrane of six mice per group are shown. *Indicates tumor nodule. ( c ) The histogram of quantified weight of metastatic nodules >8 mm 3 and the number of metastatic nodules in three groups at the time of sacrifice are shown. ( d ) RT-PCR results of TIMP2 mRNA expression of the eighteen SKOV3 xenografts in three groups. ( e ) Western blot analysis of EZH2 and TIMP2 protein expression of the xenografts. Five tissues for each group are shown because one tissue in the shEZH2 group and one in the cotransfected group were too small for protein extraction. The cropped blots are used in the figure, and full-length blots are presented in Supplementary Fig. . ( f ) The hematoxylin and eosin staining and immunohistochemical analysis of xenografts. EZH2 and TIMP2 protein expressions in control and intervention groups were verified. (scale bar, 20 μm). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Then, the slides were incubated with primary antibody against EZH2 (1:200 dilution, Cell Signaling Technology, Danvers, MA, USA) and TIMP2 (1:200 dilution, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Stable Transfection, Transfection, In Vitro, In Vivo, Membrane, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Protein Extraction, Staining, Immunohistochemical staining, Control

Journal: Scientific Reports

Article Title: EZH2-mediated epigenetic silencing of TIMP2 promotes ovarian cancer migration and invasion

doi: 10.1038/s41598-017-03362-z

Figure Lengend Snippet: EZH2 inhibits TIMP2 expression via DNA methylation. ( a ) qRT-PCR and ( b ) Western blot analysis of TIMP2 expression in A2780 and SKOV3 cells after treatment with 5Aza. The cropped blots are used in the figure, and full-length blots are presented in Supplementary Fig. . ( c ) Methylation-specific PCR (MSP) analysis revealed that 5Aza treatment markedly decreased TIMP2 promoter methylation. The cropped gel is used in the figure, and the full-length gel is presented in Supplementary Fig. . M, methylated; U, unmethylated; IVD, in vitro methylated DNA; methylation index refers to the percentage of M/M + U. ( d ) qRT-PCR and ( e ) Western blot analysis of TIMP2 expression in A2780 and SKOV3 cells after DNMT1 and DNMT3A siRNA transfection. The cropped blots are used in the figure, and full-length blots are presented in Supplementary Fig. . ( f ) MSP for DNA methylation status of the TIMP2 promoter in ovarian cancer cells after DNMT1 and DNMT3A siRNA transfection. The cropped gel is used in the figure, and the full-length gel is presented in Supplementary Fig. . ( g ) MSP for DNA methylation status of the TIMP2 promoter in the EZH2-regulated ovarian cancer cells. The cropped gels are used in the figure, and full-length gels are presented in Supplementary Fig. . ( h ) qRT-PCR and ( i ) Western blot analysis of TIMP2 expression in A2780 and SKOV3 cells after treatment with GSK126. The cropped blots are used in the figure, and full-length blots are presented in Supplementary Fig. . ( j ) MSP analysis of TIMP2 promoter methylation in A2780 and SKOV3 cells after treatment with GSK126. The cropped gels are used in the figure, and full-length gels are presented in Supplementary Fig. . * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Then, the slides were incubated with primary antibody against EZH2 (1:200 dilution, Cell Signaling Technology, Danvers, MA, USA) and TIMP2 (1:200 dilution, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Expressing, DNA Methylation Assay, Quantitative RT-PCR, Western Blot, Methylation, In Vitro, Transfection

Journal: Scientific Reports

Article Title: EZH2-mediated epigenetic silencing of TIMP2 promotes ovarian cancer migration and invasion

doi: 10.1038/s41598-017-03362-z

Figure Lengend Snippet: EZH2 recruits DNMTs at the TIMP2 promoter. ( a ) Location of binding sites of primers for MSP and qRT-PCR following ChIP in the context of CpG islands at the TIMP2 promoter. TSS, Transcription start site. ( b ) qRT-PCR analysis of immunoprecipitated chromatin by EZH2 and H3K27me3 antibodies. IgG served as a negative control. ( c ) A2780 and ( d ) OV2008 cells were transfected with EZH2 overexpression plasmid or control followed by ChIP and qRT–PCR for the quantitative detection of EZH2, H3K27me3, DNMT1 and DNMT3a on the TIMP2 promoter. The results were normalized to the input. * P < 0.05; ** P < 0.01; *** P < 0.001. NS, not significant.

Article Snippet: Then, the slides were incubated with primary antibody against EZH2 (1:200 dilution, Cell Signaling Technology, Danvers, MA, USA) and TIMP2 (1:200 dilution, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Binding Assay, Quantitative RT-PCR, Immunoprecipitation, Negative Control, Transfection, Over Expression, Plasmid Preparation, Control

Journal: Scientific Reports

Article Title: EZH2-mediated epigenetic silencing of TIMP2 promotes ovarian cancer migration and invasion

doi: 10.1038/s41598-017-03362-z

Figure Lengend Snippet: Inhibition of EZH2 converts histone modification mark occupancy at the TIMP2 promoter. ( a ) Western blot analysis of different histone modification marks in A2780 and SKOV3 cells after EZH2 knockdown or pharmacological inhibition by GSK126 treatment ( b ). The cropped blots are used in the figure, and full-length blots are presented in Supplementary Figs and . ( c ) ChIP analysis of different histone modification marks at the TIMP2 promoter in A2780 and SKOV3 cells. IgG served as a negative control. ( d , e ) ChIP analysis of different histone modification marks at the TIMP2 promoter in A2780 and SKOV3 cells treated by GSK126. ( f , g ) ChIP analysis of different histone modification marks in EZH2-knockdown A2780 and SKOV3 cells. The results were normalized to the input. The EZH2 target gene KLF2 was used as a positive control. * P < 0.05; ** P < 0.01; *** P < 0.001. NS, not significant.

Article Snippet: Then, the slides were incubated with primary antibody against EZH2 (1:200 dilution, Cell Signaling Technology, Danvers, MA, USA) and TIMP2 (1:200 dilution, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Inhibition, Modification, Western Blot, Knockdown, Negative Control, Positive Control

Journal: Reproductive Sciences

Article Title: Endometrial miR-200c is Altered During Transformation into Cancerous States and Targets the Expression of ZEBs , VEGFA , FLT1 , IKKβ , KLF9 , and FBLN5

doi: 10.1177/1933719112438448

Figure Lengend Snippet: Relative expression of miR-200c, ZEB1, and ZEB2, in endometrial biopsies (A) from mid-late proliferative phase (MLP), early- (ES), mid- (MS), and late (LS)-secretory phase of the menstrual cycle and endometrial tissues (B) from proliferative (Pro) and secretory (Sec) phases of the menstrual cycle, peri- and postmenopausal period (PPM), women exposed to GnRH agonist (GnRHa) and Depo-Provera (Depo), and grade I, II, and III endometrial cancer (C). The results are presented as mean ± standard error of the mean (SEM) and analyzed using analysis of variance (ANOVA) or nonparametric student t test (*P < .05 as compared to levels in mid-late proliferative phase [MLP] in Figure A, proliferative phase [Pro] in Figure B and peri- and postmenopausal period [PPM] in Figure C).

Article Snippet: Aliquots of 30 μg of total protein were subjected to Western blot analysis and transferred to polyvinylidene difluoride (PVDF) membrane, and then the membranes were probed with primary antibodies (dilution:1:200) generated against ZEB1, ZEB2, VEGFA, FLT1, FBLN5, TIMP2, IKKβ, and KLF9 (Cell Signaling Technology, Danvers, Massachusetts; Abcam Inc, San Francisco, California; Santa Cruz Biotechnology, Santa Cruz, California). α-tubulin (1:2000) was used for normalization and loading control.

Techniques: Expressing

Journal: Reproductive Sciences

Article Title: Endometrial miR-200c is Altered During Transformation into Cancerous States and Targets the Expression of ZEBs , VEGFA , FLT1 , IKKβ , KLF9 , and FBLN5

doi: 10.1177/1933719112438448

Figure Lengend Snippet: Relative expression of miR-200c, ZEB1, and ZEB2 in Ishikawa cells treated with 17-β Estradiol (E2) (A), progesterone (P4) (B), or medroxy progesterone acetate (MPA) (C) for 6 and 24 hours as compared to untreated control (Ctrl). The results are presented as mean ± standard error of the mean (SEM) and analyzed by analysis of variance (ANOVA) and nonparametric student t test (*P < .05 from their respective Ctrl).

Article Snippet: Aliquots of 30 μg of total protein were subjected to Western blot analysis and transferred to polyvinylidene difluoride (PVDF) membrane, and then the membranes were probed with primary antibodies (dilution:1:200) generated against ZEB1, ZEB2, VEGFA, FLT1, FBLN5, TIMP2, IKKβ, and KLF9 (Cell Signaling Technology, Danvers, Massachusetts; Abcam Inc, San Francisco, California; Santa Cruz Biotechnology, Santa Cruz, California). α-tubulin (1:2000) was used for normalization and loading control.

Techniques: Expressing, Control

Journal: Reproductive Sciences

Article Title: Endometrial miR-200c is Altered During Transformation into Cancerous States and Targets the Expression of ZEBs , VEGFA , FLT1 , IKKβ , KLF9 , and FBLN5

doi: 10.1177/1933719112438448

Figure Lengend Snippet: Relative expression of ZEB1 (A), ZEB2 (B), and CDH1 (C), in Ishikawa cells transfected with pre-miR-200c or pre-miR negative control (NC) determined by real-time polymerase chain reaction (PCR). The results are presented as mean ± standard error of the mean (SEM) and analyzed using nonparametric student t test with P values indicated by corresponding lines.

Article Snippet: Aliquots of 30 μg of total protein were subjected to Western blot analysis and transferred to polyvinylidene difluoride (PVDF) membrane, and then the membranes were probed with primary antibodies (dilution:1:200) generated against ZEB1, ZEB2, VEGFA, FLT1, FBLN5, TIMP2, IKKβ, and KLF9 (Cell Signaling Technology, Danvers, Massachusetts; Abcam Inc, San Francisco, California; Santa Cruz Biotechnology, Santa Cruz, California). α-tubulin (1:2000) was used for normalization and loading control.

Techniques: Expressing, Transfection, Negative Control, Real-time Polymerase Chain Reaction

Journal: Reproductive Sciences

Article Title: Endometrial miR-200c is Altered During Transformation into Cancerous States and Targets the Expression of ZEBs , VEGFA , FLT1 , IKKβ , KLF9 , and FBLN5

doi: 10.1177/1933719112438448

Figure Lengend Snippet: Western blot analysis of ZEB1, CDH1, VEGFA, IKKβ, KLF9, FLT1 and FBLN5 in Ishikawa cells transfected with pre-miR-200c and pre-miR negative control (NC) with α-Tubulin serving as loading control.

Article Snippet: Aliquots of 30 μg of total protein were subjected to Western blot analysis and transferred to polyvinylidene difluoride (PVDF) membrane, and then the membranes were probed with primary antibodies (dilution:1:200) generated against ZEB1, ZEB2, VEGFA, FLT1, FBLN5, TIMP2, IKKβ, and KLF9 (Cell Signaling Technology, Danvers, Massachusetts; Abcam Inc, San Francisco, California; Santa Cruz Biotechnology, Santa Cruz, California). α-tubulin (1:2000) was used for normalization and loading control.

Techniques: Western Blot, Transfection, Negative Control, Control

Journal: Reproductive Sciences

Article Title: Endometrial miR-200c is Altered During Transformation into Cancerous States and Targets the Expression of ZEBs , VEGFA , FLT1 , IKKβ , KLF9 , and FBLN5

doi: 10.1177/1933719112438448

Figure Lengend Snippet: Firefly luciferase assay with pZEX-MT01 or pGL3-Luc constructs carrying a 3′ untranslated region (3′ UTR) fragment of ZEB1 (A), ZEB2 (B), VEGFA (C), IKKβ (D), and KLF9 (E), respectively. Ishikawa cells were co-transfected with firefly luciferase reporters, Renilla luciferase transfection control plasmid (in case of IKKβ ), pre-miR-200c, or pre-miR negative control (NC). The ratio of firefly: Renilla was determined and reported as relative luciferase activity as compared to empty vector and analyzed using non-parametric student t-test with p values indicated by corresponding lines.

Article Snippet: Aliquots of 30 μg of total protein were subjected to Western blot analysis and transferred to polyvinylidene difluoride (PVDF) membrane, and then the membranes were probed with primary antibodies (dilution:1:200) generated against ZEB1, ZEB2, VEGFA, FLT1, FBLN5, TIMP2, IKKβ, and KLF9 (Cell Signaling Technology, Danvers, Massachusetts; Abcam Inc, San Francisco, California; Santa Cruz Biotechnology, Santa Cruz, California). α-tubulin (1:2000) was used for normalization and loading control.

Techniques: Luciferase, Construct, Transfection, Control, Plasmid Preparation, Negative Control, Activity Assay

Journal: Reproductive Sciences

Article Title: Endometrial miR-200c is Altered During Transformation into Cancerous States and Targets the Expression of ZEBs , VEGFA , FLT1 , IKKβ , KLF9 , and FBLN5

doi: 10.1177/1933719112438448

Figure Lengend Snippet: The miR-200c seed sequence and its complementary binding site on respective 3′ untranslated region (UTR) of its predicated target genes (represented in bold letters)

Article Snippet: Aliquots of 30 μg of total protein were subjected to Western blot analysis and transferred to polyvinylidene difluoride (PVDF) membrane, and then the membranes were probed with primary antibodies (dilution:1:200) generated against ZEB1, ZEB2, VEGFA, FLT1, FBLN5, TIMP2, IKKβ, and KLF9 (Cell Signaling Technology, Danvers, Massachusetts; Abcam Inc, San Francisco, California; Santa Cruz Biotechnology, Santa Cruz, California). α-tubulin (1:2000) was used for normalization and loading control.

Techniques: Sequencing, Binding Assay