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Proteintech rabbit polyclonal anti upf1
Rabbit Polyclonal Anti Upf1, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+anti+upf1/UPF1+Antibody/pmc08684126-85-20-25
Average 93 stars, based on 34 article reviews

rabbit polyclonal anti upf1 - by Bioz Stars, 2026-09

93/100 stars

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Article Title: Long Noncoding RNA PVT1 Promotes Breast Cancer Proliferation and Metastasis by Binding mIR-128-3p and UPF1
Article Snippet: The primary antibodies were: rabbit monoclonal anti-E-cadherin (#3195, 1:1000, Cell Signaling Technology), rabbit monoclonal anti-Vimentin (#5741, 1:1000, Cell Signaling Technology), rabbit polyclonal anti-UPF1 (23379-1-AP, 1:1000, ProteinTech), rabbit polyclonal anti-FOXQ1 (23718-1-AP, 1:1000, ProteinTech), mouse monoclonal anti-β-actin (A5316, 1:5000, Sigma-Aldrich), and mouse monoclonal anti-PCNA (sc25280, 1:1000, Santa Cruz).

Article Title: Long noncoding RNA PVT1 promotes breast cancer proliferation and metastasis by binding miR-128-3p and UPF1
Article Snippet: The primary antibodies were: rabbit monoclonal anti-E-cadherin (#3195, 1:1000, Cell Signaling Technology), rabbit monoclonal anti-Vimentin (#5741, 1:1000, Cell Signaling Technology), rabbit polyclonal anti-UPF1 (23379-1-AP, 1:1000, ProteinTech), rabbit polyclonal anti-FOXQ1 (23718-1-AP, 1:1000, ProteinTech), mouse monoclonal anti-β-actin (A5316, 1:5000, Sigma-Aldrich), and mouse monoclonal anti-PCNA (sc-25280, 1:1000, Santa Cruz).

Image Search Results

Primer sequences used for the SyberGreen RT-qPCR experiments.

Journal: Biomedicines

Article Title: The NMD Pathway Regulates GABARAPL1 mRNA during the EMT

doi: 10.3390/biomedicines9101302

Figure Lengend Snippet: Primer sequences used for the SyberGreen RT-qPCR experiments.

Article Snippet: For the Western blotting experiments and the IHC, the following antibodies were used: rabbit polyclonal anti- GABARAPL1 (D5R9Y, Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti- UPF1 (D15G6, Cell Signaling), rabbit polyclonal antibody anti-SMG1 (Q25, Cell Signaling), rabbit polyclonal anti-b-actin (A5060, Sigma Aldrich, St. Louis, MI, USA), monoclonal anti-Vimentin (#7902917, Ventana Medical Systems, Oro Valley, AZ, USA), secondary goat polyclonal anti-rabbit HRP (BI2407, Abliance, Compiègne, France) and anti-mouse HRP (BI2413C, Abliance).

Techniques:

Immunohistochemical analysis of GABARAPL1 and UPF1 in both EMT+ and EMT- NSCLCs. ( a ) Representative examples of GABARAPL1 and UPF1 immunolabelled tissues. NSCLC specimens were divided according to their VIMENTIN immunoreactivity (EMT+, n = 20 versus EMT−, n = 22). ( b ) Semi-quantitative evaluation of GABARAPL1 and UPF1 expression (intensity × extent) in NSCLCs. The mean ± S.E.M. (plus each data point) are represented. p -Values were calculated using Student’s t -test. **: p ≤ 0.01.

Journal: Biomedicines

Article Title: The NMD Pathway Regulates GABARAPL1 mRNA during the EMT

doi: 10.3390/biomedicines9101302

Figure Lengend Snippet: Immunohistochemical analysis of GABARAPL1 and UPF1 in both EMT+ and EMT- NSCLCs. ( a ) Representative examples of GABARAPL1 and UPF1 immunolabelled tissues. NSCLC specimens were divided according to their VIMENTIN immunoreactivity (EMT+, n = 20 versus EMT−, n = 22). ( b ) Semi-quantitative evaluation of GABARAPL1 and UPF1 expression (intensity × extent) in NSCLCs. The mean ± S.E.M. (plus each data point) are represented. p -Values were calculated using Student’s t -test. **: p ≤ 0.01.

Techniques: Immunohistochemical staining, Expressing

Expression of NMD and autophagy factors during the EMT. A549 cells were treated for 6, 15 or 48 h using TGFβ (5 ng/mL) and TNFα (20 ng/mL) to induce the EMT. ( a ) Quantification using RT-qPCR of mRNA expression levels of NMD and ( b ) autophagy factors. The values were calculated using the ΔΔCT method, normalized to the H3B2 levels and expressed as fold changes. Data are represented as mean ± S.E.M. of three independent experiments. p -Values were calculated using Student’s t -test. ( c ) Western blotting analysis of UPF1 expression during the TGFβ/TNFα treatment. ( d ) Quantification using Western blotting of UPF1 expression levels. Protein levels were quantified using Image Lab and Stain-Free was used as the loading control. Data are represented as mean ± S.E.M. of three independent experiments. p -Values were calculated using Student’s t -test. ns: Not significant, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001 and ****: p ≤ 0.0001.

Journal: Biomedicines

Article Title: The NMD Pathway Regulates GABARAPL1 mRNA during the EMT

doi: 10.3390/biomedicines9101302

Figure Lengend Snippet: Expression of NMD and autophagy factors during the EMT. A549 cells were treated for 6, 15 or 48 h using TGFβ (5 ng/mL) and TNFα (20 ng/mL) to induce the EMT. ( a ) Quantification using RT-qPCR of mRNA expression levels of NMD and ( b ) autophagy factors. The values were calculated using the ΔΔCT method, normalized to the H3B2 levels and expressed as fold changes. Data are represented as mean ± S.E.M. of three independent experiments. p -Values were calculated using Student’s t -test. ( c ) Western blotting analysis of UPF1 expression during the TGFβ/TNFα treatment. ( d ) Quantification using Western blotting of UPF1 expression levels. Protein levels were quantified using Image Lab and Stain-Free was used as the loading control. Data are represented as mean ± S.E.M. of three independent experiments. p -Values were calculated using Student’s t -test. ns: Not significant, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001 and ****: p ≤ 0.0001.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Control

Regulation of the ATG8 family mRNAs’ expression upon siRNA-mediated inhibition of the NMD. A549 cells were transfected with siRNA (20 pmol/well) targeting NMD transcripts UPF1 or SMG1 . ( a ) Quantification using RT-qPCR of ATG8 transcripts expression levels. The values were calculated using the ΔΔCT method, normalized to H3B2 levels and expressed as fold changes. Data are represented as mean ± S.E.M. of three independent experiments. p -Values were calculated using Student’s t -test. ( b ) Quantification using Western blotting of GABARAPL1 , UPF1 and SMG1 expression levels. ( c ) Protein levels were quantified using Image Lab and Stain-Free was used as the loading control. Data are represented as mean ± S.E.M. of three independent experiments. p -Values were calculated using Student’s t -test. ns: Not significant, *: p ≤ 0.05, **: p ≤ 0.01 and ****: p ≤ 0.0001.

Journal: Biomedicines

Article Title: The NMD Pathway Regulates GABARAPL1 mRNA during the EMT

doi: 10.3390/biomedicines9101302

Figure Lengend Snippet: Regulation of the ATG8 family mRNAs’ expression upon siRNA-mediated inhibition of the NMD. A549 cells were transfected with siRNA (20 pmol/well) targeting NMD transcripts UPF1 or SMG1 . ( a ) Quantification using RT-qPCR of ATG8 transcripts expression levels. The values were calculated using the ΔΔCT method, normalized to H3B2 levels and expressed as fold changes. Data are represented as mean ± S.E.M. of three independent experiments. p -Values were calculated using Student’s t -test. ( b ) Quantification using Western blotting of GABARAPL1 , UPF1 and SMG1 expression levels. ( c ) Protein levels were quantified using Image Lab and Stain-Free was used as the loading control. Data are represented as mean ± S.E.M. of three independent experiments. p -Values were calculated using Student’s t -test. ns: Not significant, *: p ≤ 0.05, **: p ≤ 0.01 and ****: p ≤ 0.0001.

Techniques: Expressing, Inhibition, Transfection, Quantitative RT-PCR, Western Blot, Staining, Control

Analysis of the mRNAs bound to UPF1 by RNA-immunoprecipitation. A549 cells (10 million cells) were harvested and a UPF1 RNA-immunoprecipitation was conducted. ( a ) The UPF1 protein recovery rate was quantified using Western blotting in comparison to a 10% input. Data are represented as mean ± S.E.M. of three independent experiments. ( b ) Quantification of mRNAs bound to UPF1 using RT-qPCR. The values were calculated using the ΔCT method and were relative to the 10% of the input and expressed as a fold change. Data are represented as mean ± S.E.M. of three independent experiments. p -Values were calculated using Student’s t -test. ns: Not significant, *: p ≤ 0.05, **: p ≤ 0.01.

Journal: Biomedicines

Article Title: The NMD Pathway Regulates GABARAPL1 mRNA during the EMT

doi: 10.3390/biomedicines9101302

Figure Lengend Snippet: Analysis of the mRNAs bound to UPF1 by RNA-immunoprecipitation. A549 cells (10 million cells) were harvested and a UPF1 RNA-immunoprecipitation was conducted. ( a ) The UPF1 protein recovery rate was quantified using Western blotting in comparison to a 10% input. Data are represented as mean ± S.E.M. of three independent experiments. ( b ) Quantification of mRNAs bound to UPF1 using RT-qPCR. The values were calculated using the ΔCT method and were relative to the 10% of the input and expressed as a fold change. Data are represented as mean ± S.E.M. of three independent experiments. p -Values were calculated using Student’s t -test. ns: Not significant, *: p ≤ 0.05, **: p ≤ 0.01.

Techniques: RNA Immunoprecipitation, Western Blot, Comparison, Quantitative RT-PCR

Schematic representation of eIF4A3 and UPF1 binding sites on the 3′UTR of GABARAP , GABARAPL1 and LC3B . The experimental binding sites of eIF4A3 and UPF1 were studied using the AURA 2.5.2 database.

Journal: Biomedicines

Article Title: The NMD Pathway Regulates GABARAPL1 mRNA during the EMT

doi: 10.3390/biomedicines9101302

Figure Lengend Snippet: Schematic representation of eIF4A3 and UPF1 binding sites on the 3′UTR of GABARAP , GABARAPL1 and LC3B . The experimental binding sites of eIF4A3 and UPF1 were studied using the AURA 2.5.2 database.

Techniques: Binding Assay

PIWIL1 cooperates with UPF1 to negatively regulate PIWIL1-bound RNAs in a piRNA-independent manner. (A) A Coomassie blue staining protein gel of PIWIL1-co-immunoprecipitation, Mass spectrometry of this protein gel identifies PIWIL1 (blue arrow) as well as UPF1 (red arrow). (B) Western blotting showing reciprocal co-immunoprecipitated between PIWIL1 and UPF1 (total and phosphorylated form, p-UPF1) and between PIWIL1 and UPF2. (C) Western blotting shows that PIWIL1 co-immunoprecipitates with NMD complex core proteins UPF1, phosphorylated UPF1 (p-UPF1), UPF2, and SMG1. (D) Immunofluorescence staining of PIWIL1 (green) and DCP1A (red, a P body marker) in wildtype and PIWIL_KO SNU-1 cells. (E) Immunofluorescence staining of PIWIL1 (green), DCP1A (red), and UPF1 (fuchsia) in SNU-1 cells, which shows PIWIL1 co-localized with UPF1 in the P body. (F) Venn diagram of PIWIL1-bound RNAs, UPF1-bound RNAs, and PIWIL1-negatively regulated RNAs, with P<0.05 cutoff and fold-change>=1.5 cutoff. 203 PIWIL1-negatively regulated RNAs are targeted by both PIWIL1 and UPF1. (G) Volcano plot of PIWIL1 negatively regulated direct-targets (blue dots) in SNU-1 cells. Orange dots are PIWIL1 negatively regulated direct-targets, which are also targeted by UPF1. Dots demarcated by black outlines are six cancer cell migration-related turquoise module hub genes (VCL, FLNA, LAMC3, SRCIN1, TPM2, and MYO18B). Dotted lines of the Volcano plot represent 1.5-fold-change in expression (vertical lines) and P < 0.01 cutoff (horizontal line). (H) Quantitative RIP-PCR confirmed that the six cancer cell migration-related turquoise module hub genes are bound by UPF1. (I) KEGG pathway analysis of 203 PIWIL1-negatively regulated RNAs that are targeted by both PIWIL1 and UPF1. (J) Docking model of PIWIL1 protein structure (yellow), UPF1 protein structure (green), and RNA (orange). Schematic of full length-PIWIL1 and UPF1-interacting domain mutants-PIWIL1. (K) Co-immunoprecipitation mapping of the UPF1-interacting domain of PIWIL1. pcDNA-Flag: empty vector, Flag-PIWIL1-FL: full-length PIWIL1, Flag-PIWIL1-ΔN: Flag-tagged PIWIL1 without 251-383 amino acids, Flag-PIWIL1-ΔC: Flag-tagged PIWIL1 without 624-758 amino acids, Flag-PIWIL1-ΔNΔC: Flag-tagged PIWIL1 without both 251-383 and 624-758 amino acids. (L) Western blotting of co-immunoprecipitation showing that piRNA-binding mutant PIWIL1 (Flag-piRNA-BM-PIWIL1) interacts with UPF1 in the same way as WT PIWIL1 (Flag-PIWIL1-FL). (M) Quantitative RT-PCR showing that the upregulation of PIWIL1-UPF1 co-targeted mRNAs in PIWIL1-KO cells can be rescued by WT-PIWIL1 but not the mutant PIWIL1 lacking the UPF1-interacting domain (KO-ΔNΔC-PIWIL1). (N) Transwell assays showing that the inhibition of cell migration in PIWIL1-KO cells can be rescued by WT-PIWIL1 but not the mutant PIWIL1 lacking the UPF1-interacting domain (KO-ΔNΔC-PIWIL1). (O) The bar graph shows the numbers of migrated cells of each view in the Transwell assay.

Journal: bioRxiv

Article Title: PIWIL1 Promotes Gastric Cancer via a piRNA-Independent Mechanism

doi: 10.1101/2020.05.03.075390

Figure Lengend Snippet: PIWIL1 cooperates with UPF1 to negatively regulate PIWIL1-bound RNAs in a piRNA-independent manner. (A) A Coomassie blue staining protein gel of PIWIL1-co-immunoprecipitation, Mass spectrometry of this protein gel identifies PIWIL1 (blue arrow) as well as UPF1 (red arrow). (B) Western blotting showing reciprocal co-immunoprecipitated between PIWIL1 and UPF1 (total and phosphorylated form, p-UPF1) and between PIWIL1 and UPF2. (C) Western blotting shows that PIWIL1 co-immunoprecipitates with NMD complex core proteins UPF1, phosphorylated UPF1 (p-UPF1), UPF2, and SMG1. (D) Immunofluorescence staining of PIWIL1 (green) and DCP1A (red, a P body marker) in wildtype and PIWIL_KO SNU-1 cells. (E) Immunofluorescence staining of PIWIL1 (green), DCP1A (red), and UPF1 (fuchsia) in SNU-1 cells, which shows PIWIL1 co-localized with UPF1 in the P body. (F) Venn diagram of PIWIL1-bound RNAs, UPF1-bound RNAs, and PIWIL1-negatively regulated RNAs, with P<0.05 cutoff and fold-change>=1.5 cutoff. 203 PIWIL1-negatively regulated RNAs are targeted by both PIWIL1 and UPF1. (G) Volcano plot of PIWIL1 negatively regulated direct-targets (blue dots) in SNU-1 cells. Orange dots are PIWIL1 negatively regulated direct-targets, which are also targeted by UPF1. Dots demarcated by black outlines are six cancer cell migration-related turquoise module hub genes (VCL, FLNA, LAMC3, SRCIN1, TPM2, and MYO18B). Dotted lines of the Volcano plot represent 1.5-fold-change in expression (vertical lines) and P < 0.01 cutoff (horizontal line). (H) Quantitative RIP-PCR confirmed that the six cancer cell migration-related turquoise module hub genes are bound by UPF1. (I) KEGG pathway analysis of 203 PIWIL1-negatively regulated RNAs that are targeted by both PIWIL1 and UPF1. (J) Docking model of PIWIL1 protein structure (yellow), UPF1 protein structure (green), and RNA (orange). Schematic of full length-PIWIL1 and UPF1-interacting domain mutants-PIWIL1. (K) Co-immunoprecipitation mapping of the UPF1-interacting domain of PIWIL1. pcDNA-Flag: empty vector, Flag-PIWIL1-FL: full-length PIWIL1, Flag-PIWIL1-ΔN: Flag-tagged PIWIL1 without 251-383 amino acids, Flag-PIWIL1-ΔC: Flag-tagged PIWIL1 without 624-758 amino acids, Flag-PIWIL1-ΔNΔC: Flag-tagged PIWIL1 without both 251-383 and 624-758 amino acids. (L) Western blotting of co-immunoprecipitation showing that piRNA-binding mutant PIWIL1 (Flag-piRNA-BM-PIWIL1) interacts with UPF1 in the same way as WT PIWIL1 (Flag-PIWIL1-FL). (M) Quantitative RT-PCR showing that the upregulation of PIWIL1-UPF1 co-targeted mRNAs in PIWIL1-KO cells can be rescued by WT-PIWIL1 but not the mutant PIWIL1 lacking the UPF1-interacting domain (KO-ΔNΔC-PIWIL1). (N) Transwell assays showing that the inhibition of cell migration in PIWIL1-KO cells can be rescued by WT-PIWIL1 but not the mutant PIWIL1 lacking the UPF1-interacting domain (KO-ΔNΔC-PIWIL1). (O) The bar graph shows the numbers of migrated cells of each view in the Transwell assay.

Article Snippet: 50 μL of empty Dynabeads ® Protein A were washed by Citrate-Phosphate Buffer (pH 5.0) and then incubated with 15ul Rabbit Monoclonal anti-UPF1 antibody (Cell Signaling TECHNOLOGY, Cat# 12040) or 5ug Rabbit polyclonal anti-phospho-Upf1 (Ser1127) antibody (MERCK, Cat# 07-1016) or 5ug Rabbit polyclonal anti-UPF2 antibody (Abcam, ab157108) or 5ug Normal Rabbit IgG Polyclonal Antibody (MERCK, Cat# 12-370).

Techniques: Staining, Immunoprecipitation, Mass Spectrometry, Western Blot, Immunofluorescence, Marker, Migration, Expressing, Plasmid Preparation, Binding Assay, Mutagenesis, Quantitative RT-PCR, Inhibition, Transwell Assay

a Co-immunoprecipitation (co-IP) analysis of HDFs with anti-CSL antibodies or non-immune IgG followed by immunoblotting with antibodies against the indicated proteins. n (strain) = 2. b Sequential co-IP analysis of HDFs with antibodies against CSL followed by IP with antibodies against UPF1 or non-immune IgG, and immunoblotting with antibodies against the indicated proteins. n (strain) = 3. c Proximity ligation assays (PLAs) of CSL and UPF1 association. HDFs with silenced CSL were used as negative control. Scale bar, 2 μm. Number of dots per cell was counted. n (cells) > 43 per condition, n (strain) = 3, * p < 0.05, two-tailed unpaired t -test. d PLAs of CSL and Ku70 and Ku80 association. Scale bar, 2 μm. Number of dots per cell was counted. n (cells) > 54 per condition, n (strain) = 3. e Binding of recombinant CSL and Ku70 proteins as measured by microscale thermophoresis (MST). Inset: thermophoretic movement of fluorescently-labeled CSL. Specificity controls are in Supplementary Fig. . f RT-qPCR of CAF effector genes in HDFs plus/minus UPF1/Ku70/Ku80 versus CSL gene silencing for 6 days. Silencing controls are in Supplementary Fig. . g RNA-seq analysis of CAF effector genes in HDFs plus/minus UPF1 versus CSL gene silencing for 7 days. Heatmap of differentially expressed genes in HDFs with CSL or UPF1 silencing relative to control is in log2 scale. Bars represent mean ± SD

Journal: Nature Communications

Article Title: CSL controls telomere maintenance and genome stability in human dermal fibroblasts

doi: 10.1038/s41467-019-11785-7

Figure Lengend Snippet: a Co-immunoprecipitation (co-IP) analysis of HDFs with anti-CSL antibodies or non-immune IgG followed by immunoblotting with antibodies against the indicated proteins. n (strain) = 2. b Sequential co-IP analysis of HDFs with antibodies against CSL followed by IP with antibodies against UPF1 or non-immune IgG, and immunoblotting with antibodies against the indicated proteins. n (strain) = 3. c Proximity ligation assays (PLAs) of CSL and UPF1 association. HDFs with silenced CSL were used as negative control. Scale bar, 2 μm. Number of dots per cell was counted. n (cells) > 43 per condition, n (strain) = 3, * p < 0.05, two-tailed unpaired t -test. d PLAs of CSL and Ku70 and Ku80 association. Scale bar, 2 μm. Number of dots per cell was counted. n (cells) > 54 per condition, n (strain) = 3. e Binding of recombinant CSL and Ku70 proteins as measured by microscale thermophoresis (MST). Inset: thermophoretic movement of fluorescently-labeled CSL. Specificity controls are in Supplementary Fig. . f RT-qPCR of CAF effector genes in HDFs plus/minus UPF1/Ku70/Ku80 versus CSL gene silencing for 6 days. Silencing controls are in Supplementary Fig. . g RNA-seq analysis of CAF effector genes in HDFs plus/minus UPF1 versus CSL gene silencing for 7 days. Heatmap of differentially expressed genes in HDFs with CSL or UPF1 silencing relative to control is in log2 scale. Bars represent mean ± SD

Article Snippet: Antibodies used were anti-rabbit γ-H2AX antibody (Cat. 2577, Cell Signaling, 1:100 dilution), anti-goat VIMENTIN polyclonal antibody (Cat. AF2105, R&D, 1:200 dilution), anti-mouse VIMENTIN monoclonal antibody (Cat. 20346, Abcam, 1:200 dilution), anti-mouse CD45 monoclonal antibody (Cat. 304001, Biolegend, 1:200 dilution), anti-mouse CSL monoclonal antibody (Cat. 271128, Santa Cruz, 1:50 dilution), anti-rabbit UPF1 polyclonal antibody (Cat. HPA019587, Sigma, 1:100 dilution), and anti-mouse PDGFRα-FITC monoclonal antibody (Cat. 21789, Santa Cruz, 1:50 dilution).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Ligation, Negative Control, Two Tailed Test, Binding Assay, Recombinant, Microscale Thermophoresis, Labeling, Quantitative RT-PCR, RNA Sequencing Assay

a Telomere binding assay by ChIP/qPCR with antibodies against CSL and UPF1, individually or sequentially (CSL+UPF1), in parallel with non-immune IgG, in HDFs as in , . n (strain) = 3, * p < 0.05, two-tailed unpaired t -test. b Telomere binding assays by ChIP/qPCR with antibodies against the indicated proteins in HDFs (GB1) plus/minus siRNA-mediated CSL silencing (3 days) or CSL silencing and concomitant lentivirally induced CSL overexpression (OE). Similar experiments with two additional HDF strains (GB3 and GB4) are in Supplementary Fig. . c Telomere binding assays by ChIP/qPCR analysis of the same cells as in b with antibodies against TRF1/TRF2. Similar experiments with two additional HDF strains (GB3 and GB4) are in Supplementary Fig. . d Telomere binding assays by ChIP/qPCR with antibodies against the indicated proteins in HDFs (GB1) plus/minus lentivirally induced CSL overexpression (OE) for 3 days. Similar experiments with two additional HDF strains (GB3 and GB4) are in Supplementary Fig. . e Telomere binding assays by ChIP/qPCR with anti FLAG-tag antibodies in HEK293T cells expressing increasing amounts (0, 250 ng, 500 ng, and 2 μg) of FLAG-tagged full length (FL) CSL together with full length (FL) Ku70 (2 μg). Non-immune IgGs were used for normalization. A second independent experiment is in Supplementary Fig. . f PLAs of stromal fibroblasts (identified by VIMENTIN staining) from unaffected skin versus flanking SCC with TRF1 or TRF2 antibodies in combination with antibodies against the other indicated proteins. Scale bar, 5 μm. Quantification of CSL and UPF1/Ku70/Ku80/TRF1/TRF2 levels in the same samples are in Fig. and Supplementary Fig. , respectively. Triangles, circles, and squares point to values from flanking skin (black) and corresponding SCC (red) from three patients. Non-immune IgGs were used as control. Mean ± SD, n (cells) > 77 per condition, n (SCC) = 3, n (matched Skin) = 3, * p < 0.05, two-tailed paired t -test. Bars represent mean ± SD

Journal: Nature Communications

Article Title: CSL controls telomere maintenance and genome stability in human dermal fibroblasts

doi: 10.1038/s41467-019-11785-7

Figure Lengend Snippet: a Telomere binding assay by ChIP/qPCR with antibodies against CSL and UPF1, individually or sequentially (CSL+UPF1), in parallel with non-immune IgG, in HDFs as in , . n (strain) = 3, * p < 0.05, two-tailed unpaired t -test. b Telomere binding assays by ChIP/qPCR with antibodies against the indicated proteins in HDFs (GB1) plus/minus siRNA-mediated CSL silencing (3 days) or CSL silencing and concomitant lentivirally induced CSL overexpression (OE). Similar experiments with two additional HDF strains (GB3 and GB4) are in Supplementary Fig. . c Telomere binding assays by ChIP/qPCR analysis of the same cells as in b with antibodies against TRF1/TRF2. Similar experiments with two additional HDF strains (GB3 and GB4) are in Supplementary Fig. . d Telomere binding assays by ChIP/qPCR with antibodies against the indicated proteins in HDFs (GB1) plus/minus lentivirally induced CSL overexpression (OE) for 3 days. Similar experiments with two additional HDF strains (GB3 and GB4) are in Supplementary Fig. . e Telomere binding assays by ChIP/qPCR with anti FLAG-tag antibodies in HEK293T cells expressing increasing amounts (0, 250 ng, 500 ng, and 2 μg) of FLAG-tagged full length (FL) CSL together with full length (FL) Ku70 (2 μg). Non-immune IgGs were used for normalization. A second independent experiment is in Supplementary Fig. . f PLAs of stromal fibroblasts (identified by VIMENTIN staining) from unaffected skin versus flanking SCC with TRF1 or TRF2 antibodies in combination with antibodies against the other indicated proteins. Scale bar, 5 μm. Quantification of CSL and UPF1/Ku70/Ku80/TRF1/TRF2 levels in the same samples are in Fig. and Supplementary Fig. , respectively. Triangles, circles, and squares point to values from flanking skin (black) and corresponding SCC (red) from three patients. Non-immune IgGs were used as control. Mean ± SD, n (cells) > 77 per condition, n (SCC) = 3, n (matched Skin) = 3, * p < 0.05, two-tailed paired t -test. Bars represent mean ± SD

Techniques: Binding Assay, Two Tailed Test, Over Expression, FLAG-tag, Expressing, Staining

a Co-IP analysis of HEK293T cells expressing MYC-tagged full length (FL) Ku70 and 1-257, 1-464, 1-573, or 257–609 domains plus/minus full length (FL) CSL with anti-MYC magnetic beads followed by immunoblotting with antibodies against the indicated proteins. b Co-IP analysis of HEK293T cells expressing MYC-tagged full length (FL) Ku70 or its 464–609 domain plus/minus full length (FL) CSL with anti-MYC magnetic beads followed by immunoblotting with antibodies against the indicated proteins. c Co-IP analysis of HEK293T cells expressing FLAG-tagged full length (FL) CSL and CSL BTD (166–334) domains plus/minus full length (FL) UPF1 with anti-FLAG magnetic beads followed by immunoblotting with antibodies against the indicated proteins. A second independent experiment is in Supplementary Fig. . d Co-IP analysis of HEK293T cells expressing FLAG-tagged full length (FL) CSL and CSL point mutants (R192H, F235R, V237R, A258R, and Q307R) plus/minus full length (FL) Ku70 with anti-FLAG magnetic beads followed by immunoblotting with antibodies against the indicated proteins. Additional information on CSL point mutants is in Supplementary Fig. . e Cartoon representation showing the docking complex between CSL (cyan)—Telomere DNA (orange)—Ku70 (magenta) and Ku80 (blue) using HDOCK server. Ku70 is shown to interact with CSL-BTD domain and bind to CSL bound telomere DNA through its SAP domain, while Ku80 binds indirectly through Ku70. f Close up view of the docking complex between CSL—Telomere DNA—Ku70 showing the α-helix of C-Ku domain (olive) interacting with CSL-BTD domain (cyan), and Ku70 SAP domain (magenta) interacting with telomere DNA (orange). The “hot spot” mutations that abrogate CSL-Ku70 interaction and Ku70-telomeric DNA association as in d are shown in pink (A258R and R192H). Additional non-interfering mutations are labeled in blue (F235R, V237R, and Q307R). g – j Telomeric binding assays with antibodies against the indicated proteins followed by qPCR with telomere- and alu-specific primers in HEK293T cells expressing CSL full length (WT) and point mutants (R192H, F235R, V237R, A258R, and Q307R). Non-immune IgGs were used for normalization. Bars represent mean ± SD

Journal: Nature Communications

Article Title: CSL controls telomere maintenance and genome stability in human dermal fibroblasts

doi: 10.1038/s41467-019-11785-7

Figure Lengend Snippet: a Co-IP analysis of HEK293T cells expressing MYC-tagged full length (FL) Ku70 and 1-257, 1-464, 1-573, or 257–609 domains plus/minus full length (FL) CSL with anti-MYC magnetic beads followed by immunoblotting with antibodies against the indicated proteins. b Co-IP analysis of HEK293T cells expressing MYC-tagged full length (FL) Ku70 or its 464–609 domain plus/minus full length (FL) CSL with anti-MYC magnetic beads followed by immunoblotting with antibodies against the indicated proteins. c Co-IP analysis of HEK293T cells expressing FLAG-tagged full length (FL) CSL and CSL BTD (166–334) domains plus/minus full length (FL) UPF1 with anti-FLAG magnetic beads followed by immunoblotting with antibodies against the indicated proteins. A second independent experiment is in Supplementary Fig. . d Co-IP analysis of HEK293T cells expressing FLAG-tagged full length (FL) CSL and CSL point mutants (R192H, F235R, V237R, A258R, and Q307R) plus/minus full length (FL) Ku70 with anti-FLAG magnetic beads followed by immunoblotting with antibodies against the indicated proteins. Additional information on CSL point mutants is in Supplementary Fig. . e Cartoon representation showing the docking complex between CSL (cyan)—Telomere DNA (orange)—Ku70 (magenta) and Ku80 (blue) using HDOCK server. Ku70 is shown to interact with CSL-BTD domain and bind to CSL bound telomere DNA through its SAP domain, while Ku80 binds indirectly through Ku70. f Close up view of the docking complex between CSL—Telomere DNA—Ku70 showing the α-helix of C-Ku domain (olive) interacting with CSL-BTD domain (cyan), and Ku70 SAP domain (magenta) interacting with telomere DNA (orange). The “hot spot” mutations that abrogate CSL-Ku70 interaction and Ku70-telomeric DNA association as in d are shown in pink (A258R and R192H). Additional non-interfering mutations are labeled in blue (F235R, V237R, and Q307R). g – j Telomeric binding assays with antibodies against the indicated proteins followed by qPCR with telomere- and alu-specific primers in HEK293T cells expressing CSL full length (WT) and point mutants (R192H, F235R, V237R, A258R, and Q307R). Non-immune IgGs were used for normalization. Bars represent mean ± SD

Techniques: Co-Immunoprecipitation Assay, Expressing, Magnetic Beads, Western Blot, Labeling, Binding Assay

Fig. 1 Cloning strategy for the production of the pCCL.shRNA-UPF1. WPRE lentiviral vector. The pRS-shRNA-UPF1 construct was subjected to enzymatic digestion by the restriction enzymes NaeI and SalI with the purpose to isolate the 410 bp insert containing U6 promoter and UPF1 shRNA.pCCL.PGW vector, as the result of cutting by the SalI and EcoRV endonucleases, was deleted of a region containing the GFP gene and the relative PGK promoter. That region was replaced by the 410 bp insert deriving from the pRS-shRNA-UPF1, obtaining the final vector pCCL.shRNA-UPF1.WPRE

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 1 Cloning strategy for the production of the pCCL.shRNA-UPF1. WPRE lentiviral vector. The pRS-shRNA-UPF1 construct was subjected to enzymatic digestion by the restriction enzymes NaeI and SalI with the purpose to isolate the 410 bp insert containing U6 promoter and UPF1 shRNA.pCCL.PGW vector, as the result of cutting by the SalI and EcoRV endonucleases, was deleted of a region containing the GFP gene and the relative PGK promoter. That region was replaced by the 410 bp insert deriving from the pRS-shRNA-UPF1, obtaining the final vector pCCL.shRNA-UPF1.WPRE

Article Snippet: Membranes were washed three times for 5 min each with 30 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with primary rabbit monoclonal antibody against UPF1 (1:15.000) (Origene, Rockville, MD,) in 15 ml primary antibody dilution buffer (TBS, 0.1% Tween20, 5% BSA) with gentle agitation over-night at 4 °C.

Techniques: Cloning, shRNA, Plasmid Preparation, Construct

Fig. 2 Quantification of the insertion of pCCL.shRNA-UPF1.WPRE and pCCL.shRNA-scramble.WPRE vectors in the obtained clones. Analysis of the integration of pCCL.shRNA-UPF1.WPRE vector in β039.m5 (a) and βwt.wt3 (c) derived clones and of pCCL.shRNA-scramble.WPRE construct in β039.m5 (b) and βwt.wt3 (d) clones. Histograms show the integration of the lentiviral vectors understood as fold compared to insertions already present in the respective original cells, used as reference samples. The data represent the averages ± SD of three independent determinations

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 2 Quantification of the insertion of pCCL.shRNA-UPF1.WPRE and pCCL.shRNA-scramble.WPRE vectors in the obtained clones. Analysis of the integration of pCCL.shRNA-UPF1.WPRE vector in β039.m5 (a) and βwt.wt3 (c) derived clones and of pCCL.shRNA-scramble.WPRE construct in β039.m5 (b) and βwt.wt3 (d) clones. Histograms show the integration of the lentiviral vectors understood as fold compared to insertions already present in the respective original cells, used as reference samples. The data represent the averages ± SD of three independent determinations

Techniques: shRNA, Clone Assay, Plasmid Preparation, Derivative Assay, Construct

Fig. 3 Quantification of the UPF1 mRNA content in UPF1- and scramble clones. Histograms show the UPF1 mRNA content in UPF1- (a) and scramble (b) β039.m5 clones and UPF1- (c) and scramble (d) βwt.wt3 clones and in their original cell lines, β039.m5 and βwt.wt3, used as reference samples. The data were obtained by Real Time qRT-PCR using expression assays for UPF1 and GAPDH cDNAs, the last of which was used for the normalization. The data represent the averages ± SD of three independent determinations

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 3 Quantification of the UPF1 mRNA content in UPF1- and scramble clones. Histograms show the UPF1 mRNA content in UPF1- (a) and scramble (b) β039.m5 clones and UPF1- (c) and scramble (d) βwt.wt3 clones and in their original cell lines, β039.m5 and βwt.wt3, used as reference samples. The data were obtained by Real Time qRT-PCR using expression assays for UPF1 and GAPDH cDNAs, the last of which was used for the normalization. The data represent the averages ± SD of three independent determinations

Techniques: Clone Assay, Quantitative RT-PCR, Expressing

Fig. 4 Quantification of the β globin mRNA content in UPF1- and scramble β039.m5 clones. Histograms show the UPF1 mRNA content in UPF1- clone 3, scramble clone a and in the original cell line β039.m5, used as reference samples. The data were obtained by Real Time qRT-PCR using expression assays for β globin and GAPDH cDNAs, the last of which was used for the normalization. The data represent the averages ± SD of three independent determinations

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 4 Quantification of the β globin mRNA content in UPF1- and scramble β039.m5 clones. Histograms show the UPF1 mRNA content in UPF1- clone 3, scramble clone a and in the original cell line β039.m5, used as reference samples. The data were obtained by Real Time qRT-PCR using expression assays for β globin and GAPDH cDNAs, the last of which was used for the normalization. The data represent the averages ± SD of three independent determinations

Techniques: Clone Assay, Quantitative RT-PCR, Expressing

Fig. 5 Evaluation of the content of UPF1 protein in UPF1- clones. The UPF1 protein accumulation in UPF1- clones 3 (a) and 1 (b) compared to the respective control cells, β039.m5 and βwt.wt3, was determined by Western Blotting using scalar quantities of protein extracts and monoclonal antibodies against UPF1 and p70 proteins. The image obtained was analysed by densitometry, which evaluated the relative amount of protein staining and quantified the results in terms of optical density: the histogram shows the reduction in UPF1 protein content in clone 3 and 1, meant as a percentage compared to the corresponding control cells (c). Values were normalized using p70 protein. The data represent the averages ± SD of three independent determinations

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 5 Evaluation of the content of UPF1 protein in UPF1- clones. The UPF1 protein accumulation in UPF1- clones 3 (a) and 1 (b) compared to the respective control cells, β039.m5 and βwt.wt3, was determined by Western Blotting using scalar quantities of protein extracts and monoclonal antibodies against UPF1 and p70 proteins. The image obtained was analysed by densitometry, which evaluated the relative amount of protein staining and quantified the results in terms of optical density: the histogram shows the reduction in UPF1 protein content in clone 3 and 1, meant as a percentage compared to the corresponding control cells (c). Values were normalized using p70 protein. The data represent the averages ± SD of three independent determinations

Techniques: Clone Assay, Control, Western Blot, Bioprocessing, Staining

Fig. 6 Effects of geneticin (G418) on the β globin production in UPF1- clone 3, scramble clone a and relative original cell line β039.m5. After a 3 day incubation with 400 ng/μl G418, cells were labeled with the monoclonal antibody Hemoglobin β-PE and analysed by FACS. Histogram a shows the proportion of β globin producing cells and the fold of that proportion in β039.m5, clone 3 and clone a cells, untreated and treated with 400 ng/μl G418. Histogram b shows the fluorescence median of all the samples and its shift after treatment. The data represent the averages ± SD of three independent experiments

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 6 Effects of geneticin (G418) on the β globin production in UPF1- clone 3, scramble clone a and relative original cell line β039.m5. After a 3 day incubation with 400 ng/μl G418, cells were labeled with the monoclonal antibody Hemoglobin β-PE and analysed by FACS. Histogram a shows the proportion of β globin producing cells and the fold of that proportion in β039.m5, clone 3 and clone a cells, untreated and treated with 400 ng/μl G418. Histogram b shows the fluorescence median of all the samples and its shift after treatment. The data represent the averages ± SD of three independent experiments

Techniques: Incubation, Labeling, Fluorescence

Fig. 7 Effects of geneticin (G418) on the β globin production in UPF1- clone 1, scramble clone c and relative original cell line βwt.wt3. After a 3 day incubation with 400 ng/μl G418, cells were labelled with the monoclonal antibody Hemoglobin β-PE and analysed by FACS. Histogram A shows the proportion of β globin producing cells and the fold of that proportion in K562.βwt.wt3, clone 1 and clone c cells, untreated and treated with 400 ng/μl G418. Histogram B shows the fluorescence median of all the samples and its shift after treatment. The data represent the averages ± SD of three independent experiments

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 7 Effects of geneticin (G418) on the β globin production in UPF1- clone 1, scramble clone c and relative original cell line βwt.wt3. After a 3 day incubation with 400 ng/μl G418, cells were labelled with the monoclonal antibody Hemoglobin β-PE and analysed by FACS. Histogram A shows the proportion of β globin producing cells and the fold of that proportion in K562.βwt.wt3, clone 1 and clone c cells, untreated and treated with 400 ng/μl G418. Histogram B shows the fluorescence median of all the samples and its shift after treatment. The data represent the averages ± SD of three independent experiments

Techniques: Incubation, Fluorescence

Fig. 8 Effects of geneticin (G418) on the content of β globin mRNA in β039 and βwt clones. Histograms show the β globin mRNA content in β039 (a) and βwt (b) clones, UPF1 or scramble silenced, and in the original cell lines, β039.m5 and βwt.wt3 respectively, untreated and treated with 400 ng/μl G418. The data were obtained by Real Time qRT-PCR using expression assays for β globin and GAPDH cDNAs, the last of which was used for the normalization. The data represent the averages ± SD of three independent experiments

Journal: BMC biotechnology

Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β 0 39 thalassemia point mutation.

doi: 10.1186/s12896-018-0435-0

Figure Lengend Snippet: Fig. 8 Effects of geneticin (G418) on the content of β globin mRNA in β039 and βwt clones. Histograms show the β globin mRNA content in β039 (a) and βwt (b) clones, UPF1 or scramble silenced, and in the original cell lines, β039.m5 and βwt.wt3 respectively, untreated and treated with 400 ng/μl G418. The data were obtained by Real Time qRT-PCR using expression assays for β globin and GAPDH cDNAs, the last of which was used for the normalization. The data represent the averages ± SD of three independent experiments

Techniques: Clone Assay, Quantitative RT-PCR, Expressing

( a ) Western blots monitoring phosphorylation levels of endogenous UPF1 in HeLa tet-off cells transfected with indicated siRNAs followed by IP for UPF1 and anti-phospho-[S/T]Q (P-UPF1) and anti-UPF1 (UPF1) western blotting. Indicated phosphorylation levels were calculated from at least three independent experiments by dividing the P -UPF1 signal with that from anti-UPF1 (UPF1) and are shown normalized to control (LUC) conditions±s.e.m. P values are indicated below panels and are relative to control conditions (paired two-tailed Student's t -test). ( b ) Same as in a in HeLa tet-off cells transfected with myc-tagged UPF1 wild-type (‘WT'), ATP-hydrolysis mutant (DE636/637AA) or ATP-binding mutants (K498A, G495R and G497E). Indicated phosphorylation levels were calculated as in a and are shown normalized to control (‘WT') conditions±s.e.m. ( c ) Same as in a in HeLa tet-off cells transfected with indicated siRNAs. UPF1 phosphorylation levels were quantified as in a . ( d ) Same as in a in HeLa tet-off cells transfected with plasmids coding for myc-tagged UPF1 together with an empty vector (none) or vectors expressing CAF1B DDAA or DCP2 E148Q. UPF1 phosphorylation levels were quantified as in a and are shown normalized to control (‘none') conditions±s.e.m.

Journal: Nature Communications

Article Title: Hyperphosphorylation amplifies UPF1 activity to resolve stalls in nonsense-mediated mRNA decay

doi: 10.1038/ncomms12434

Figure Lengend Snippet: ( a ) Western blots monitoring phosphorylation levels of endogenous UPF1 in HeLa tet-off cells transfected with indicated siRNAs followed by IP for UPF1 and anti-phospho-[S/T]Q (P-UPF1) and anti-UPF1 (UPF1) western blotting. Indicated phosphorylation levels were calculated from at least three independent experiments by dividing the P -UPF1 signal with that from anti-UPF1 (UPF1) and are shown normalized to control (LUC) conditions±s.e.m. P values are indicated below panels and are relative to control conditions (paired two-tailed Student's t -test). ( b ) Same as in a in HeLa tet-off cells transfected with myc-tagged UPF1 wild-type (‘WT'), ATP-hydrolysis mutant (DE636/637AA) or ATP-binding mutants (K498A, G495R and G497E). Indicated phosphorylation levels were calculated as in a and are shown normalized to control (‘WT') conditions±s.e.m. ( c ) Same as in a in HeLa tet-off cells transfected with indicated siRNAs. UPF1 phosphorylation levels were quantified as in a . ( d ) Same as in a in HeLa tet-off cells transfected with plasmids coding for myc-tagged UPF1 together with an empty vector (none) or vectors expressing CAF1B DDAA or DCP2 E148Q. UPF1 phosphorylation levels were quantified as in a and are shown normalized to control (‘none') conditions±s.e.m.

Article Snippet: Lysates were incubated for 4 h with 50 μl of protein A sepharose CL-4B beads (GE Healthcare Life Sciences) conjugated to 2.5 μl of rabbit polyclonal anti-UPF1 antibody or 2.5 μl of mouse monoclonal anti-Myc (Cell Signaling, clone 9B11).

Techniques: Western Blot, Phospho-proteomics, Transfection, Control, Two Tailed Test, Mutagenesis, Binding Assay, Plasmid Preparation, Expressing

( a ) Two-dimensional gel electrophoresis assays performed on extracts from HeLa tet-off cells transfected with siRNAs targeting Firefly Luciferase (siLUC), SMG6 (siSMG6) or XRN1 (siXRN1) and subjected to western blotting with anti-UPF1 antibody. Prior to electrophoresis, cell lysates were treated (+ λ phosphatase; right panel) or not (left panel) with lambda protein phosphatase. Graphs indicate signal intensity along the blots normalized to the total signal intensity. ( b ) Two-dimensional gel electrophoresis assays performed on extracts from HeLa tet-off cells transfected with plasmids coding for myc-tagged wild-type UPF1 (Upf1-WT) or UPF1 DE636/637AA. The graph indicates signal intensity along the blots normalized to the total signal intensity.

Journal: Nature Communications

Article Title: Hyperphosphorylation amplifies UPF1 activity to resolve stalls in nonsense-mediated mRNA decay

doi: 10.1038/ncomms12434

Figure Lengend Snippet: ( a ) Two-dimensional gel electrophoresis assays performed on extracts from HeLa tet-off cells transfected with siRNAs targeting Firefly Luciferase (siLUC), SMG6 (siSMG6) or XRN1 (siXRN1) and subjected to western blotting with anti-UPF1 antibody. Prior to electrophoresis, cell lysates were treated (+ λ phosphatase; right panel) or not (left panel) with lambda protein phosphatase. Graphs indicate signal intensity along the blots normalized to the total signal intensity. ( b ) Two-dimensional gel electrophoresis assays performed on extracts from HeLa tet-off cells transfected with plasmids coding for myc-tagged wild-type UPF1 (Upf1-WT) or UPF1 DE636/637AA. The graph indicates signal intensity along the blots normalized to the total signal intensity.

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Transfection, Luciferase, Western Blot

( a ) Western blots showing endogenous UPF1 phosphorylation levels in RAW 264.7 cells treated with LPS and monitored as in . Numbers refer to hours after LPS addition. Graph showing UPF1 phosphorylation levels quantified as in is shown below. Indicated values are normalized to the zero time point±s.e.m. from three independent experiments. P values are relative to time zero and were calculated using paired two-tailed Student's t -test. ( b ) Western blots showing UPF1 phosphorylation levels in NIH 3T3 cells transfected with siRNAs targeting either Firefly Luciferase (LUC) or ZFP36, ZFP36L1 and ZFP36L2 (ZFP36/L1/L2). Numbers above panels refer to time after serum induction; lower panel shows uninduced cells. Graph showing UPF1 phosphorylation levels measured from three independent experiments as in a is shown below. P value is comparing siLUC to siZFP36/L1/L2 conditions at 1 h after serum induction using the paired two-tailed Student's t -test.

Journal: Nature Communications

Article Title: Hyperphosphorylation amplifies UPF1 activity to resolve stalls in nonsense-mediated mRNA decay

doi: 10.1038/ncomms12434

Figure Lengend Snippet: ( a ) Western blots showing endogenous UPF1 phosphorylation levels in RAW 264.7 cells treated with LPS and monitored as in . Numbers refer to hours after LPS addition. Graph showing UPF1 phosphorylation levels quantified as in is shown below. Indicated values are normalized to the zero time point±s.e.m. from three independent experiments. P values are relative to time zero and were calculated using paired two-tailed Student's t -test. ( b ) Western blots showing UPF1 phosphorylation levels in NIH 3T3 cells transfected with siRNAs targeting either Firefly Luciferase (LUC) or ZFP36, ZFP36L1 and ZFP36L2 (ZFP36/L1/L2). Numbers above panels refer to time after serum induction; lower panel shows uninduced cells. Graph showing UPF1 phosphorylation levels measured from three independent experiments as in a is shown below. P value is comparing siLUC to siZFP36/L1/L2 conditions at 1 h after serum induction using the paired two-tailed Student's t -test.

Techniques: Western Blot, Phospho-proteomics, Two Tailed Test, Transfection, Luciferase

( a ) Schematic representation of human UPF1 protein showing the amino acids mutated in this study (black); the position of the helicase domain and the cysteine/histidine-rich domain (CH) of UPF1 are indicated (not to scale). Numbers following amino-acid symbols correspond to the position in the UPF1 primary sequence (GenBank #NP_002902). Asterisk indicates confirmed phosphorylated sites. [S/T]Q motifs not investigated in this work are shown in grey. ( b ) Northern blots showing the decay of β39 mRNA in HeLa Tet-Off cells depleted for endogenous UPF1 and expressing myc-tagged variants of UPF1 as indicated. Numbers above panels refer to minutes after tetracycline-mediated transcriptional shutoff of β39 mRNA (chase). β39 mRNA half-lives ( t 1/2 ) were calculated after normalization of levels of β39 mRNA to levels of constitutively transcribed β-globin–GAPDH fusion mRNA (βWT-GAP) and are given as averages±s.e.m. from three independent experiments. Numbers on the right refer to RNA lengths in nucleotides (nts) excluding polyA-tails. ( c ) Graph showing half-lives calculated from experiments presented in b . Lower and upper dotted lines represent half-life values for wild-type UPF1 (Upf1-wt) and no add-back (None) conditions respectively. Error bars represent s.e.m. from three independent experiments. P values were calculated relative to Upf1-wt conditions using the paired two-tailed Student's t -test; * P <0.05 and ** P <0.01.

Journal: Nature Communications

Article Title: Hyperphosphorylation amplifies UPF1 activity to resolve stalls in nonsense-mediated mRNA decay

doi: 10.1038/ncomms12434

Figure Lengend Snippet: ( a ) Schematic representation of human UPF1 protein showing the amino acids mutated in this study (black); the position of the helicase domain and the cysteine/histidine-rich domain (CH) of UPF1 are indicated (not to scale). Numbers following amino-acid symbols correspond to the position in the UPF1 primary sequence (GenBank #NP_002902). Asterisk indicates confirmed phosphorylated sites. [S/T]Q motifs not investigated in this work are shown in grey. ( b ) Northern blots showing the decay of β39 mRNA in HeLa Tet-Off cells depleted for endogenous UPF1 and expressing myc-tagged variants of UPF1 as indicated. Numbers above panels refer to minutes after tetracycline-mediated transcriptional shutoff of β39 mRNA (chase). β39 mRNA half-lives ( t 1/2 ) were calculated after normalization of levels of β39 mRNA to levels of constitutively transcribed β-globin–GAPDH fusion mRNA (βWT-GAP) and are given as averages±s.e.m. from three independent experiments. Numbers on the right refer to RNA lengths in nucleotides (nts) excluding polyA-tails. ( c ) Graph showing half-lives calculated from experiments presented in b . Lower and upper dotted lines represent half-life values for wild-type UPF1 (Upf1-wt) and no add-back (None) conditions respectively. Error bars represent s.e.m. from three independent experiments. P values were calculated relative to Upf1-wt conditions using the paired two-tailed Student's t -test; * P <0.05 and ** P <0.01.

Techniques: Sequencing, Northern Blot, Expressing, Two Tailed Test

$( a ) Western blots showing protein levels in UPF1-immunoprecipitated (IP) or total (1% of Input) fractions from HeLa tet-off cells transfected with the indicated siRNAs. ( b , c ) Same as panel a but monitoring IPs and input samples for exogenously expressed Myc-tagged wild-type and mutant UPF1 proteins (8ST>A=[S/T]7,8,9,10,11,17,18,19A; 12ST>A=[S/T]1,2,7,8,9,10,11,15,16,17,18,19A). FLAG-tagged SMG6 was co-transfected with Myc-UPF1 in c . ( d ) Western blots for in vitro pull-downs of bacterially expressed UPF1 HD-SQ with FLAG-tagged GFP, SMG5/7 or SMG6 immuno-isolated from human cells. UPF1 HD-SQ was treated with or without ATM kinase prior to pull-down. Input samples are shown below. *Antibody heavy chain.$

Journal: Nature Communications

Article Title: Hyperphosphorylation amplifies UPF1 activity to resolve stalls in nonsense-mediated mRNA decay

doi: 10.1038/ncomms12434

Figure Lengend Snippet: ( a ) Western blots showing protein levels in UPF1-immunoprecipitated (IP) or total (1% of Input) fractions from HeLa tet-off cells transfected with the indicated siRNAs. ( b , c ) Same as panel a but monitoring IPs and input samples for exogenously expressed Myc-tagged wild-type and mutant UPF1 proteins (8ST>A=[S/T]7,8,9,10,11,17,18,19A; 12ST>A=[S/T]1,2,7,8,9,10,11,15,16,17,18,19A). FLAG-tagged SMG6 was co-transfected with Myc-UPF1 in c . ( d ) Western blots for in vitro pull-downs of bacterially expressed UPF1 HD-SQ with FLAG-tagged GFP, SMG5/7 or SMG6 immuno-isolated from human cells. UPF1 HD-SQ was treated with or without ATM kinase prior to pull-down. Input samples are shown below. *Antibody heavy chain.

Techniques: Western Blot, Immunoprecipitation, Transfection, Mutagenesis, In Vitro, Isolation

( a ) Northern blots showing the decay of β39 mRNA in HeLa Tet-Off cells depleted for endogenous UPF1 and expressing the indicated exogenous variants of UPF1. In addition, cells were treated with siRNAs targeting SMG5 or SMG7, or as a control, Firefly Luciferase (LUC). Numbers above the panels refer to minutes after tetracycline-mediated transcriptional shutoff of β39 mRNA (chase). β39 mRNA half-lives ( t 1/2 ) were calculated from three independent experiments as described for . Numbers on the right refer to RNA lengths in nucleotides (nts) excluding polyA-tails. ( b ) Graphs showing β39 mRNA half-lives calculated from mRNA decay assays presented in a and . Graphs compare siLUC control conditions (grey) to siSMG5 (white; left graph) or siSMG7 conditions (white; right graph). Error bars represent s.e.m. from three independent experiments. P values indicated above bars compare siSMG7 and siSMG5 conditions to control siLUC. P values indicated above brackets compare siSMG7 and siSMG5 conditions between different UPF1 mutants (all P values were calculated using the paired two-tailed Student's t -test).

Journal: Nature Communications

Article Title: Hyperphosphorylation amplifies UPF1 activity to resolve stalls in nonsense-mediated mRNA decay

doi: 10.1038/ncomms12434

Figure Lengend Snippet: ( a ) Northern blots showing the decay of β39 mRNA in HeLa Tet-Off cells depleted for endogenous UPF1 and expressing the indicated exogenous variants of UPF1. In addition, cells were treated with siRNAs targeting SMG5 or SMG7, or as a control, Firefly Luciferase (LUC). Numbers above the panels refer to minutes after tetracycline-mediated transcriptional shutoff of β39 mRNA (chase). β39 mRNA half-lives ( t 1/2 ) were calculated from three independent experiments as described for . Numbers on the right refer to RNA lengths in nucleotides (nts) excluding polyA-tails. ( b ) Graphs showing β39 mRNA half-lives calculated from mRNA decay assays presented in a and . Graphs compare siLUC control conditions (grey) to siSMG5 (white; left graph) or siSMG7 conditions (white; right graph). Error bars represent s.e.m. from three independent experiments. P values indicated above bars compare siSMG7 and siSMG5 conditions to control siLUC. P values indicated above brackets compare siSMG7 and siSMG5 conditions between different UPF1 mutants (all P values were calculated using the paired two-tailed Student's t -test).

Techniques: Northern Blot, Expressing, Control, Luciferase, Two Tailed Test

When downstream decay steps are limiting, UPF1 stalls on NMD-targeted mRNPs and is progressively phosphorylated on [S/T]Q motifs. The increase in phosphorylation progressively promotes the ability of UPF1 to activate mRNA decay.

Journal: Nature Communications

Article Title: Hyperphosphorylation amplifies UPF1 activity to resolve stalls in nonsense-mediated mRNA decay

doi: 10.1038/ncomms12434

Figure Lengend Snippet: When downstream decay steps are limiting, UPF1 stalls on NMD-targeted mRNPs and is progressively phosphorylated on [S/T]Q motifs. The increase in phosphorylation progressively promotes the ability of UPF1 to activate mRNA decay.

Techniques: Phospho-proteomics

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