Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A ) Schematic representation of FLNA structure including numbering of the Ig-like domain repeats, and labelling of the actin-binding domain (ABD). The asterisks mark the repeats lacking in the FLNA variant form used in ( B ). ( B ) Pull-down assays showing direct interaction between recombinant DPP9 and recombinant FLAG tagged wt FLNA or a mutated form of FLAG-FLNA (lacking repeats 4, 9, 12, 17, 19, 21, and 23). Shown is a representative result of at least three independent experiments. ( C ) Recombinant DPP9 binds directly to GST- FLNA construct containing repeats 5–7 but not to GST-FLNA construct containing repeats 6–7. Shown is a representative result of at least three independent experiments. ( D ) Co-immunoprecipitation of endogenous FLNA with endogenous DPP9 from HeLa cells treated with different cross-linkers. Binding was observed in the presence of the sulfhydryl cross-linker DPDPB. Shown is a representative result of at least three independent experiments. To control for the specificity of the cross link, we blotted for DPP8, which did not bind to DPP9 in the presence of DPDPB ( E ) Quantification of the proximity ligation assay (in situ PLA) visualizing DPP9-FLNA interaction in HeLa cells treated with FLNA silencing oligos or non-targeting (NT) siRNAs for control shown in ( F ). The number of PLA signals per cell were quantified in a blinded manner using the Duolink ImageTool software (SIGMA). Data are represented as mean ± SEM. Signals of more than 130 cells were quantified for each condition respectively. Statistical analysis was carried out by an unpaired two-tailed t test (***p<0.0005). ( F ) PLA showing interaction of DPP9 with FLNA in HeLa cells. Each red dot represents a single FLNA-DPP9 interaction. The number of PLA signals is significantly decreased in cells silenced for FLNA compared to cells treated with NT siRNA. Actin filaments are stained in green, nuclei were visualized by using HOECHST. Shown are representative images of at least three independent PLA experiments. DOI: http://dx.doi.org/10.7554/eLife.16370.003

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Binding Assay, Variant Assay, Recombinant, Construct, Immunoprecipitation, Control, Proximity Ligation Assay, In Situ, Software, Two Tailed Test, Staining

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A ) In vitro cleavage of a synthetic Syk peptide corresponding to the N-terminus of Syk (1–31) by recombinant DPP9. 50 µM of a synthetic Syk (1–31) peptide was incubated for 6 hr, either alone or with 130 nM DPP9. For control 10 µM allosteric DPP9 inhibitor SLRFLYEG was added in addition to 130 nM DPP9 and (6 hr). An additional control included the peptide and the inactive DPP9 S730G variant. Samples were analysed by high resolution liquid chromatography/tandem mass spectrometry in triplicate. Quantitation was achieved by extracting ion chromatograms and integrating peak areas for the most abundant 3+ charge state of the intact 1–31 ([M+3H] 3+ m/z 1149.8589) and the cleaved 3–31 ([M+3H] 3+ m/z 1082.4997) peptides. The identities and retention times of the peptides were established by accurate mass measurement and product ion spectra (data not shown). ( B – G ) PLA assays showing that the interaction between DPP9 and Syk requires the active site of DPP9. Shown are representative images with the corresponding quantifications of at least three independent PLA experiments. Actin filaments are stained in green, and nuclei were visualized by using HOECHST. The number of PLA signals (red dots) per cell were quantified in a blinded manner using the Duolink ImageTool software (SIGMA). Signals of more than 300 cells were quantified for each condition respectively. Statistical analysis was carried out by an unpaired two-tailed t test (**p<0.005; ***p<0.0005; n.s = not significant). ( B ) The interaction between DPP9 and Syk is markedly decreased in HeLa cells treated with 10 µM SLRFLYEG compared to control cells treated with DMSO. ( C ) Quantification of the PLA DPP9-Syk shown in ( B ). Data are represented as mean ± SEM. ( D ) The number of PLA signals representing DPP9-Syk interactions per cell is reduced upon treatment of HeLa cells with the competitive DPP8/9 inhibitor 1G244 (10 µM, for 5 min) compared to control cells treated with DMSO. ( E ) Quantification of the PLA DPP9-Syk shown in ( D ). Data are represented as mean ± SEM. ( F ) The interaction of DPP9 with FLNA is not significantly altered upon treatment of HeLa cells with 1G244 (10 µM, 30 min) compared to control cells treated with DMSO. ( G ) Quantification of the PLA DPP9- FLNA shown in ( F ). Data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.16370.008

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: In Vitro, Recombinant, Incubation, Control, Variant Assay, Liquid Chromatography, Mass Spectrometry, Quantitation Assay, Mass Measurement, Staining, Software, Two Tailed Test

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: HeLa cells were treated with 10 µM DPP8/9 inhibitor 1G244 or DMSO for control (0, 5 and 30 min). Cells were lysed and extracts (5 µg) of were analysed for DPP activity in the presence of the artificial DPP substrate GP-AMC (250 µM) or the unrelated substrate R-AMC (50 µM). Fluorescence was measured over time. Experiment was performed at least three times, each time in triplicates. Shown is a representative, data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.16370.009

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Control, Activity Assay, Fluorescence

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A ) Total cell lysates (10 µg per lane) of DG-75 cells stimulated with 12 µg/ml F(ab’) 2 fragment goat-anti-human IgG+IgM (0, 1 and 4 min) were analyzed for DPP9 protein levels by Western blotting. Tubulin was used as loading control. Shown is a representative blot, an experiment was performed more than five times. ( B ) DG-75 cells were treated with 10 µM DPP8/9 inhibitor 1G244 or DMSO for control (0, 5 and 30 min). Cell lysates (10 µg) of were analysed for DPP activity in the presence of the artificial DPP substrate GP-AMC (250 µM) or the unrelated substrate R-AMC (50 µM). Fluorescence was measured over time. An experiment was performed at least three times, each time in triplicates. Shown is a representative, data are represented as mean ± SEM. ( C ) Indirect immunofluorescence images of DG-75 cells decorated with antibodies against DPP9, DPP8 and DPPIV. DOI: http://dx.doi.org/10.7554/eLife.16370.013

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Western Blot, Control, Activity Assay, Fluorescence, Immunofluorescence

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A and B ) PLA showing that the interactions of FLNA with Syk and DPP9 are conserved in human DG-75 B cells. Each PLA interaction is shown here as a white dot, nuclei were visualized by using HOECHST. Control reactions (NCtrl) were performed with only one primary antibody (αSyk, αFLNA or αDPP9). Shown are representative images and quantifications of at least three independent PLA experiments. The number of PLA signals per cell were quantified in a blinded manner using the Duolink ImageTool software (SIGMA). Signals of more than 80 cells were quantified for each condition respectively. Data are represented as mean ± SEM. Statistical analysis was carried out by an unpaired two-tailed t test (***p<0.0001). ( C and D ) PLA in DG-75 cells showing that Syk interacts specifically with DPP9 but not with its homologs DPP8 and DPPIV. Control reactions (NCtrl) cells were treated with one primary antibody only: αDPP9, αDPP8 or αDPPIV. Shown are quantifications of the PLA DPP9-Syk, DPP8-Syk and DPPIV-Syk in DG-75 cells from three independent experiments. Data are represented as mean ± SEM. The number of PLA signals per cell were quantified in a blinded manner using the Duolink ImageTool software (SIGMA). Signals of more than 100 cells were quantified for each condition respectively. Statistical analysis was carried out by an unpaired two-tailed t test (***p<0.0001; n.s = not significant). ( E ) CHX chase experiment showing reduced stability of endogenous Syk upon stimulation of the BCR. Human DG-75 cells were stimulated with 12 µg/ml F(ab’) 2 fragment goat-anti-human IgG+IgM (+ stim), or left untreated (- stim), and simultaneously subjected to CHX chase. DPP9 was analysed as a loading control. Shown is one representative result of at least three independent experiments. ( F ) CHX chase experiments showing that the stability of endogenous Syk in stimulated DG-75 cells, is determined by the proteasome and by DPP9. DG-75 cells were treated either with the DPP8/9 inhibitor 1G244 (10 µM), with the proteasome inhibitor MG132 (100 µM) or with DMSO for control (MOCK). Cell lysates were analysed for protein levels of Syk and of DPP9 for loading control by Western blotting. Shown is one representative result of at least three independent experiments. ( G ) Quantification of the Western blot results shown in ( F ). The ratio of Syk/DPP9 at time 0 hr was normalized to 100%. For signal quantification GelQuant.NET software provided by biochemlabsolutions.com was used. ( H ) CHX chase experiment assaying the stability of endogenous phosphorylated Syk (p-Y323) in stimulated DG-75 cells upon treatment with the DPP8/9 inhibitor 1G244 (10 µM) or with DMSO for control (MOCK). Tubulin was assayed as loading control. Shown is one representative result of at least three independent experiments. ( I ) Quantification of the Western blot results shown in ( H ). The ratio of Syk p-Y323/tubulin at time 10 min was normalized to 100%. For signal quantification GelQuant.NET software provided by biochemlabsolutions.com was used. DOI: http://dx.doi.org/10.7554/eLife.16370.012

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Control, Software, Two Tailed Test, Western Blot

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A ) Higher levels of endogenous active Syk (phosphorylated on Y352) in stimulated DG-75 cells treated with the DPP8/9 inhibitor 1G244 compared to the mock (DMSO) treated cells. 1G244 (10 µM) was added at the same time of BCR stimulation (time 0). Tubulin was assayed for loading control. Shown is a representative result of at least three independent pulse chase experiments. ( B ) Quantification of the Western blot results shown in ( A ). The ratio of Syk p-Y352/tubulin at time 10 min was normalized to 100%. For signal quantification GelQuant.NET software provided by biochemlabsolutions.com was used. ( C ) Inhibition of DPP9 in DG-75 cells leads to increased Ca 2+ mobilization, which is not dependant on BCR stimulation. Shown are flow cytometric Ca 2+ profiles after the addition of 10 µM DPP8/9 inhibitor 1G244 or DMSO for control (marked by an arrow). To monitor Ca 2+ mobilization upon BCR stimulation in either 1G244-treated or control cells, cells were treated with 10 µg/ml F(ab) 2 goat-anti-human IgM. ( D ) Same as in ( C ) using Ramos cells as a second B cell line. ( E – F ) In the absence of BCR stimulation, DPP9 inhibition leads to higher basal levels of phosphorylated Syk and its down stream effector protein PLCγ2. ( E ) Western blotting analysis of DG-75 cells treated with 1G244 in the absence of BCR stimulation. Lysates were analysed with antibodies specific against phosphorylated Syk (p-Y352) or phosphorylated PLCγ2. For loading control lysates were analysed with antibodies recognizing unmodified Syk and PLCγ2. ( F ) Cells were treated for 20 min with 1G244 (10 µM) or DMSO for control. Alternatively, cells were treated for 30 min with the allosteric DPP9 inhibitor SLRFLYEG peptide complexed with the carrier peptide (pep-1). Control cells were treated with the carrier peptide only. Following inhibitor treatment, cells were lysed and subjected to immunoprecipitation assays against Phospho-Y. Eluted proteins were analysed for Syk and PLCγ2 levels by Western blotting. Total protein levels in cell lysates were monitored for control. ( G ) Lower levels of phosphorylated ERK1/2 (both bands) are detected in the 1G244 treated DG-75 cells compared to mock (DMSO) treated cells. 1G244 (10 µM) was added prior to BCR stimulation (time 0). DPP9 was assayed for loading control. Shown is a representative result of at least three independent pulse chase experiments. ( H ) Quantification of the Western blot results shown in ( G ) as described in ( B ). DOI: http://dx.doi.org/10.7554/eLife.16370.015

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Control, Pulse Chase, Western Blot, Software, Inhibition, Immunoprecipitation

Journal: Nature Communications

Article Title: A NOTCH feed-forward loop drives reprogramming from adrenergic to mesenchymal state in neuroblastoma

doi: 10.1038/s41467-019-09470-w

Figure Lengend Snippet: NOTCH paralogues induce a mesenchymal phenotype in ADRN neuroblastoma cells. a Western blot analysis of cell lysates of SH-SY5Y with inducible overexpression of NOTCH1-IC, NOTCH2-IC FLAG or NOTCH3-IC. Immunoblots show NOTCH1-IC, NOTCH2-IC FLAG or NOTCH3 proteins as well as protein of the known NOTCH target gene HES1 to confirm induction of each transgene. Lysates are analyzed for MES-markers (FN1 and SNAI2) and ADRN-markers (PHOX2A, PHOX2B, and DBH). β-actin was used as loading control. IC intracellular domain, TM transmembrane domain, dox doxycycline. Source data are provided as a Source Data file. b Transwell migration assay of SH-SY5Y cells with inducible overexpression of NOTCH1-IC, NOTCH2-IC FLAG or NOTCH3-IC. Cells were allowed to migrate for 48 h. dox, doxycycline. Box plots show the number of migrated cells per High-Power Field (HPF). Whiskers denote the interval within 1.5 times the interquartile range (box edges) of the median (center line). Two-sided Student’s t -test assuming equal variance was used to calculate statistical difference, *** p < 0.001. Source data are provided as a Source Data file

Article Snippet: Other primary antibodies were FN1 (AF1918, R&D systems), PHOX2A (sc81978), PHOX2B (sc376997, Santa Cruz), α-tubulin (T5168, Sigma) and β-actin (ab6276, Abcam).

Techniques: Western Blot, Over Expression, Control, Transwell Migration Assay

Journal: Nature Communications

Article Title: A NOTCH feed-forward loop drives reprogramming from adrenergic to mesenchymal state in neuroblastoma

doi: 10.1038/s41467-019-09470-w

Figure Lengend Snippet: NOTCH3-IC is a master regulator of ADRN-to-MES reprogramming a . Analysis of mRNA signature scores for MES and ADRN cells in a panel of neuroblastoma cell lines and a time series of NOTCH3-IC induction. Control (-dox) SH-SY5Y-NOTCH3-IC cells are depicted in blue; SH-SY5Y cells with induction of NOTCH3-IC (+dox) are shown in red. Time of NOTCH3-IC induction is indicated in days. Dashed circles group cell lines of MES- or ADRN-type as previously determined . b Z-score mRNA expression of core super-enhancer associated transcription factors specific for ADRN or MES cells. Time of NOTCH3-IC induction (in days) is indicated above. dox, doxycycline. c Western blot analysis showing proteins of ADRN-markers (PHOX2A, PHOX2B, DBH, and TFAP2B), MES-markers (FN1, SNAI2, YAP1, and SOX9) and members of the NOTCH pathway (JAG1, NOTCH1, NOTCH2, NOTCH3, MAML2, and HES1) in SH-SY5Y with inducible overexpression of NOTCH3-IC. Time of NOTCH3-IC induction is indicated above the western blots. Protein lysates from 691-MES and 691-ADRN cells are loaded on the same blot as a reference for MES and ADRN states, respectively. β-actin was used as loading control. dox, doxycycline. Source data are provided as a Source Data file

Article Snippet: Other primary antibodies were FN1 (AF1918, R&D systems), PHOX2A (sc81978), PHOX2B (sc376997, Santa Cruz), α-tubulin (T5168, Sigma) and β-actin (ab6276, Abcam).

Techniques: Control, Expressing, Western Blot, Over Expression

Journal: Nature Communications

Article Title: A NOTCH feed-forward loop drives reprogramming from adrenergic to mesenchymal state in neuroblastoma

doi: 10.1038/s41467-019-09470-w

Figure Lengend Snippet: NOTCH-IC paralogues induce a reciprocal feed-forward signaling cascade. a SH-SY5Y cells with inducible overexpression of NOTCH3-IC were profiled for gene expression up to 21 days. Z-scores of mRNA expression values are shown for JAG1 , NOTCH1 , NOTCH2 , NOTCH3 , MAML2 , and HES1 . The transcription of NOTCH receptors from endogenous loci is measured using probesets located in the 3′UTRs of NOTCH1 (probeset 218902_at), NOTCH2 (probeset 202443_x_at) and NOTCH3 (probeset 203238_s_at). Notably, the 3′UTR is absent in the NOTCH3-IC transgene. b . Western blot analysis of NOTCH1, NOTCH2, NOTCH3, MAML2, and JAGGED1 proteins in SH-SY5Y with NOTCH1-IC, NOTCH2-IC FLAG or NOTCH3-IC inducible overexpression. β-actin was used as loading control. Time of induction was 96 h. Source data are provided as a Source Data file. c Western blot analysis of SH-SY5Y cells with (+dox) or without (−dox) NOTCH3-IC expression that were cultured in the presence (+) or the absence (−) of the gamma-secretase inhibitor RO4929097. Protein lysates were analyzed for NOTCH pathway genes (NOTCH1, MAML2, HES1), MES markers (FN1, YAP1, SNAI2) and ADRN markers (PHOX2A, PHOX2B, GATA2, TFAP2B). β-actin was used as loading control. dox, doxycycline. Source data are provided as a Source Data file. d Analysis of MES and ADRN mRNA signature scores in SH-SY5Y cells with NOTCH3-IC induction that were treated with the gamma-secretase inhibitor RO4929097. Control cells (-dox) treated with DMSO or RO4929097 are shown in dark- and light blue, respectively. NOTCH3-IC expressing cells with DMSO or RO4929097 are shown in red and purple, respectively. Cells were induced with doxycycline and treated with RO4929097 for 14 days. Dashed circles indicate groups of MES- or ADRN-type cell lines as previously determined

Article Snippet: Other primary antibodies were FN1 (AF1918, R&D systems), PHOX2A (sc81978), PHOX2B (sc376997, Santa Cruz), α-tubulin (T5168, Sigma) and β-actin (ab6276, Abcam).

Techniques: Over Expression, Gene Expression, Expressing, Western Blot, Control, Cell Culture

Journal: Nature Communications

Article Title: A NOTCH feed-forward loop drives reprogramming from adrenergic to mesenchymal state in neuroblastoma

doi: 10.1038/s41467-019-09470-w

Figure Lengend Snippet: A transient phase of NOTCH3-IC expression induces commitment to a MES phenotype. a Western blot analysis of transient NOTCH3-IC induction. Time of doxycycline (dox) induction is indicated above the blots. Induction was followed by doxycycline wash-out and all samples were harvested after T = 14 days. Immunoblots show NOTCH pathway proteins (JAG1, NOTCH1, and NOTCH3), MES-markers (FN1 and YAP1) and ADRN-markers (PHOX2A, PHOX2B, DBH, and GATA2). As a positive reference, a sample of continued NOTCH3-IC induction for T = 14 days was run on the same gel (right). β-actin was used as loading control. Source data are provided as a Source Data file. b Summed z-scores of mRNA signatures for MES and ADRN genesets. Non-induced control (−dox) cells are shown in dark blue. Persistent NOTCH3-IC induction of T = 7 and 14 days are shown in pink and red, respectively. A transient phase of NOTCH3-IC induction ( T = 7 days + doxycycline, followed by T = 7 days - doxycycline wash-out) is shown in light blue. As a reference, various neuroblastoma cell lines are shown in gray dots and grouped by dashed lines according to MES and ADRN phenotypes (see ref. ). c , d mRNA analysis (z-score of expression) of C) NOTCH pathway genes and D) core transcription factors of MES and ADRN lineages. + and—indicate culture conditions with or without doxycycline, respectively. Time of doxycycline treatment is indicated in days. For color-coding of samples, see panel c

Article Snippet: Other primary antibodies were FN1 (AF1918, R&D systems), PHOX2A (sc81978), PHOX2B (sc376997, Santa Cruz), α-tubulin (T5168, Sigma) and β-actin (ab6276, Abcam).

Techniques: Expressing, Western Blot, Control

Journal: Oncology Letters

Article Title: miR-20a-5p inhibits endometrial cancer progression by targeting janus kinase 1

doi: 10.3892/ol.2021.12688

Figure Lengend Snippet: Expression of Jak1 in human EC tissues. (A) Jak1 was upregulated in human EC tissues. (B) Jak1 mRNA expression was negatively correlated with miR-20a-5p expression in human EC tissues. *P<0.05. miR, microRNA; EC, endometrial cancer; Jak, janus kinase 1.

Article Snippet: After washing in Tris-buffered saline-Tween-20 (0.05%) solution, membranes were incubated with mouse monoclonal primary antibodies against Jak1 (1:400; cat. no. sc376996; Santa Cruz Biotechnology, Inc.) and GAPDH (1:1,000; cat. no. sc365062; Santa Cruz Biotechnology, Inc.) at 4°C overnight.

Techniques: Expressing

Journal: Oncology Letters

Article Title: miR-20a-5p inhibits endometrial cancer progression by targeting janus kinase 1

doi: 10.3892/ol.2021.12688

Figure Lengend Snippet: miR-20a-5p directly targeted Jak1. (A) Luciferase activity of wt-Jak1 3′UTR was significantly decreased in cells transfected with miR-20a-5p-mimic. (B) Effect of miR-20a-5p on Jak1 mRNA and protein expression. *P<0.05 and # P<0.01. miR, microRNA; EC, endometrial cancer; Jak, janus kinase 1; wt, wild-type; mut, mutant; NC, negative control.

Article Snippet: After washing in Tris-buffered saline-Tween-20 (0.05%) solution, membranes were incubated with mouse monoclonal primary antibodies against Jak1 (1:400; cat. no. sc376996; Santa Cruz Biotechnology, Inc.) and GAPDH (1:1,000; cat. no. sc365062; Santa Cruz Biotechnology, Inc.) at 4°C overnight.

Techniques: Luciferase, Activity Assay, Transfection, Expressing, Mutagenesis, Negative Control

Journal: Oncology Letters

Article Title: miR-20a-5p inhibits endometrial cancer progression by targeting janus kinase 1

doi: 10.3892/ol.2021.12688

Figure Lengend Snippet: Jak1 reversed the effects of miR-20a-5p on cell viability, cell invasion and adhesion. Expression of (A) Jak1 protein and (B) miR-20a-5p in Ishikawa cells following transfection with miR-20a-5p-mimic and Jak1-pcDNA3.1. Jak1 overexpression abolished the effects of miR-20a-5p-mimic on (C) cell proliferation, (D) cell invasive ability (magnification, ×400) and (E) cell adhesion. *P<0.05 and # P<0.01 vs. NC + pc DNA3.1 group; & P<0.05 and @ P<0.01 vs. miR-20a-5p + pc DNA3.1 group. miR, microRNA; EC, endometrial cancer; Jak, janus kinase 1; wt, wild-type; mut, mutant; NC, negative control.

Article Snippet: After washing in Tris-buffered saline-Tween-20 (0.05%) solution, membranes were incubated with mouse monoclonal primary antibodies against Jak1 (1:400; cat. no. sc376996; Santa Cruz Biotechnology, Inc.) and GAPDH (1:1,000; cat. no. sc365062; Santa Cruz Biotechnology, Inc.) at 4°C overnight.

Techniques: Expressing, Transfection, Over Expression, Mutagenesis, Negative Control