block it fluorescent sirna  (Thermo Fisher)


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    Name:
    BLOCK iT Fluorescent Oligo for lipid transfection
    Description:
    BLOCK iT Fluorescent Oligo provides • Strong easily detectable FITC signal indicating transfection efficiency• Chemical modifications for a clearer more persistent cellular signal than labeled siRNA• Proven correlation of transfection efficiency with Stealth RNAi siRNA and traditional siRNA moleculesThis makes the BLOCK iT Fluorescent Oligo an ideal indicator of transfection efficiency for RNAi experiments BLOCK iT Fluorescent Oligo is available in two configurations one designed for lipid mediated transfection 2 x 125 µL at 20 µM and one designed for electroporation 75 µl at 1 mM
    Catalog Number:
    2013
    Price:
    None
    Category:
    Standards Ladders Controls
    Applications:
    Cell Culture|RNAi|RNAi Transfection|RNAi, Epigenetics & Non-Coding RNA Research|Synthetic siRNA Transfection|siRNA|Transfection
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    Structured Review

    Thermo Fisher block it fluorescent sirna
    KYL-conjugated protamine increases the uptake of <t>siRNA</t> in LNCaP cells. Two and a half microliters of the <t>BLOCK-iT™</t> Fluorescent siRNA (20 μ M) were mixed with 1.5 μ g protamine (or the protamine-KYL conjugate) in 100 μ l serum-free
    BLOCK iT Fluorescent Oligo provides • Strong easily detectable FITC signal indicating transfection efficiency• Chemical modifications for a clearer more persistent cellular signal than labeled siRNA• Proven correlation of transfection efficiency with Stealth RNAi siRNA and traditional siRNA moleculesThis makes the BLOCK iT Fluorescent Oligo an ideal indicator of transfection efficiency for RNAi experiments BLOCK iT Fluorescent Oligo is available in two configurations one designed for lipid mediated transfection 2 x 125 µL at 20 µM and one designed for electroporation 75 µl at 1 mM
    https://www.bioz.com/result/block it fluorescent sirna/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    block it fluorescent sirna - by Bioz Stars, 2021-04
    95/100 stars

    Images

    1) Product Images from "Identification of a LNCaP-Specific Binding Peptide Using Phage Display"

    Article Title: Identification of a LNCaP-Specific Binding Peptide Using Phage Display

    Journal: Pharmaceutical research

    doi: 10.1007/s11095-011-0469-7

    KYL-conjugated protamine increases the uptake of siRNA in LNCaP cells. Two and a half microliters of the BLOCK-iT™ Fluorescent siRNA (20 μ M) were mixed with 1.5 μ g protamine (or the protamine-KYL conjugate) in 100 μ l serum-free
    Figure Legend Snippet: KYL-conjugated protamine increases the uptake of siRNA in LNCaP cells. Two and a half microliters of the BLOCK-iT™ Fluorescent siRNA (20 μ M) were mixed with 1.5 μ g protamine (or the protamine-KYL conjugate) in 100 μ l serum-free

    Techniques Used: Blocking Assay

    Related Articles

    Blocking Assay:

    Article Title: Gene silencing in tick cell lines using small interfering or long double-stranded RNA
    Article Snippet: .. Block-it green fluorescent siRNA (Invitrogen) was used to analyse transfection efficiency. .. Scrambled, non-targeting siRNA (Thermo Scientific) was used as a negative control.

    Article Title: Inflammasome activation in airway epithelial cells after multi-walled carbon nanotube exposure mediates a profibrotic response in lung fibroblasts
    Article Snippet: siRNA for NLRP3 Cells were transfected using Lipofectamine 2000 reagent (Block-iT Transfection Kit, Invitrogen) according to manufacturer’s recommendation and incubated with specific 100 nM siRNAs for 24 hours prior to nanomaterial exposures. .. Silencer select® siRNA against NLRP3 (sense 5′- > 3′ GCUUUGUCCUCGGUACUCAtt and UGAGUACCGAGGACAAAGCtg, Silencer select® negative control #2 siRNA (product number 4390846 Ambion®) and BLOCK-iT™ Green Fluorescent Oligo for Lipid Transfection (transfection control, Invitrogen) were used. .. Inflammasome down regulation was verified with RT- qPCR and NLRP3 protein analyses (Additional file ).

    Article Title: siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro
    Article Snippet: A scrambled sequence with the same base composition as siRNA-69 was also designed as the negative control (siRNA-SCS) and shared no significant homology with the target gene. .. A BLOCK-iT Fluorescent Oligo (Invitrogen Life Technologies, Carlsbad, CA, USA) labeled with fluorescein isothiocyanate (FITC) was synthesized to test the transfection efficiency of the siRNAs. .. All the siRNAs were synthesized by Invitrogen.

    Article Title: Nodal Signaling via an Autocrine Pathway Promotes Proliferation of Mouse Spermatogonial Stem/Progenitor Cells through Smad2/3 and Oct-4 Activation
    Article Snippet: Since we observed that type A spermatogonia from 6-day-old mice and C18-4 cells expressed Nodal, we sought to determine the effect of intrinsic Nodal expression changes on the proliferation and survival of mouse SSCs using RNAi. .. The BLOCK-iT™ fluorescent oligo has a correlation of transfection efficiency with siRNA molecules (Invitrogen). .. As shown in , transfection efficiency of Nodal siRNAs in C18-4 cells was 75 ± 2.4% (mean ± SEM, n = 3) as indicated by the uptake of the BLOCK-iT™ fluorescent oligo.

    Article Title: Identification of a LNCaP-Specific Binding Peptide Using Phage Display
    Article Snippet: N-Succinimidyl-3-(2-pyridyldithio) propionate (SPDP) was purchased from Thermo Fisher Scientific, Inc. (Pittsburgh, PA). .. The BLOCK-iT™ Fluorescent siRNA was obtained from Invitrogen (Carlsbad, CA). ..

    Article Title: Gfra1 Silencing in Mouse Spermatogonial Stem Cells Results in Their Differentiation Via the Inactivation of RET Tyrosine Kinase 1
    Article Snippet: The Stealth RNAi negative control, Gfra1 siRNAs, and the BLOCK-iT fluorescent oligo were transfected into type A spermatogonia and Sertoli cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction, with certain modifications. .. Briefly, 50 pmol Stealth RNAi negative control, Gfra1 siRNA-1, Gfra1 siRNA-2, and the BLOCK-iT fluorescent oligo were diluted in 100 μl Opti-MEM I Reduced Serum Medium (Invitrogen) and then mixed gently. .. Lipofectamine 2000 (2 μl) was diluted in 100 μl Opti-MEM I Reduced Serum Medium and incubated for 15 min at room temperature.

    Transfection:

    Article Title: Gene silencing in tick cell lines using small interfering or long double-stranded RNA
    Article Snippet: .. Block-it green fluorescent siRNA (Invitrogen) was used to analyse transfection efficiency. .. Scrambled, non-targeting siRNA (Thermo Scientific) was used as a negative control.

    Article Title: Inflammasome activation in airway epithelial cells after multi-walled carbon nanotube exposure mediates a profibrotic response in lung fibroblasts
    Article Snippet: siRNA for NLRP3 Cells were transfected using Lipofectamine 2000 reagent (Block-iT Transfection Kit, Invitrogen) according to manufacturer’s recommendation and incubated with specific 100 nM siRNAs for 24 hours prior to nanomaterial exposures. .. Silencer select® siRNA against NLRP3 (sense 5′- > 3′ GCUUUGUCCUCGGUACUCAtt and UGAGUACCGAGGACAAAGCtg, Silencer select® negative control #2 siRNA (product number 4390846 Ambion®) and BLOCK-iT™ Green Fluorescent Oligo for Lipid Transfection (transfection control, Invitrogen) were used. .. Inflammasome down regulation was verified with RT- qPCR and NLRP3 protein analyses (Additional file ).

    Article Title: siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro
    Article Snippet: A scrambled sequence with the same base composition as siRNA-69 was also designed as the negative control (siRNA-SCS) and shared no significant homology with the target gene. .. A BLOCK-iT Fluorescent Oligo (Invitrogen Life Technologies, Carlsbad, CA, USA) labeled with fluorescein isothiocyanate (FITC) was synthesized to test the transfection efficiency of the siRNAs. .. All the siRNAs were synthesized by Invitrogen.

    Article Title: miR-612 negatively regulates colorectal cancer growth and metastasis by targeting AKT2
    Article Snippet: Mutation and deletion of miR-612 binding sites in the AKT2 3′-UTR were achieved by site-directed mutagenesis using the QuikChangeH XL Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA, USA) according to the manufacturer's instructions. .. Cell transfection Oligonucleotides including miR-612 mimic and the miR-612 inhibitor anti-miR-612 were used (Thermo Scientific, Lafayette, CO, USA) for overexpression or inhibition of miR-612, respectively. .. For cell transfection assays, the synthetic oligonucleotides were transfected into cells using a Lipofectamine RNAiMAX Kit (Invitrogen) at about 50% confluence, according to the product manual.

    Article Title: Nodal Signaling via an Autocrine Pathway Promotes Proliferation of Mouse Spermatogonial Stem/Progenitor Cells through Smad2/3 and Oct-4 Activation
    Article Snippet: Since we observed that type A spermatogonia from 6-day-old mice and C18-4 cells expressed Nodal, we sought to determine the effect of intrinsic Nodal expression changes on the proliferation and survival of mouse SSCs using RNAi. .. The BLOCK-iT™ fluorescent oligo has a correlation of transfection efficiency with siRNA molecules (Invitrogen). .. As shown in , transfection efficiency of Nodal siRNAs in C18-4 cells was 75 ± 2.4% (mean ± SEM, n = 3) as indicated by the uptake of the BLOCK-iT™ fluorescent oligo.

    Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
    Article Snippet: Then, siRNAs were dissolved with Rnase free H2 O to the concentration of 20 μM. .. To determine the optimal transfection condition, various combinations of siRNA concentrations (25 nM, 50 nM and 100 nM FITC labeled nonsilencing siRNA, FITC-Oligo, Invitrogen) and transfection reagent volumes (0.5 μl, 1 μl, 1.5μl and 2 μl Lipofectamine 2000, Lipo 2000, Invitrogen) were examined. .. Before transfection, PFFs were cultured in 400 μl Opti-MEM (GIBCO), 0.5 μl, 1 μl, 1.5μl or 2 μl Lipo 2000 was added into Opti-MEM and incubated at room temperature for 5 min, 20 μM FITC-Oligo was diluted into 250 nM, 500 nM or 1000 nM with Opti-MEM, then, 100 μl FITC-Oligo-Lipo 2000 complexes was obtained through a mixture of 50 μl Opti-MEM with 250 nM, 500 nM or 1000 nM FITC-Oligo and 50 μl Opti-MEM with 0.5 μl, 1 μl, 1.5μl or 2 μl Lipo 2000, incubated at room temperature for 30 min and added into each 24-well culture plate with PFFs and 400 μl Opti-MEM.

    Negative Control:

    Article Title: Inflammasome activation in airway epithelial cells after multi-walled carbon nanotube exposure mediates a profibrotic response in lung fibroblasts
    Article Snippet: siRNA for NLRP3 Cells were transfected using Lipofectamine 2000 reagent (Block-iT Transfection Kit, Invitrogen) according to manufacturer’s recommendation and incubated with specific 100 nM siRNAs for 24 hours prior to nanomaterial exposures. .. Silencer select® siRNA against NLRP3 (sense 5′- > 3′ GCUUUGUCCUCGGUACUCAtt and UGAGUACCGAGGACAAAGCtg, Silencer select® negative control #2 siRNA (product number 4390846 Ambion®) and BLOCK-iT™ Green Fluorescent Oligo for Lipid Transfection (transfection control, Invitrogen) were used. .. Inflammasome down regulation was verified with RT- qPCR and NLRP3 protein analyses (Additional file ).

    Article Title: Gfra1 Silencing in Mouse Spermatogonial Stem Cells Results in Their Differentiation Via the Inactivation of RET Tyrosine Kinase 1
    Article Snippet: The Stealth RNAi negative control, Gfra1 siRNAs, and the BLOCK-iT fluorescent oligo were transfected into type A spermatogonia and Sertoli cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction, with certain modifications. .. Briefly, 50 pmol Stealth RNAi negative control, Gfra1 siRNA-1, Gfra1 siRNA-2, and the BLOCK-iT fluorescent oligo were diluted in 100 μl Opti-MEM I Reduced Serum Medium (Invitrogen) and then mixed gently. .. Lipofectamine 2000 (2 μl) was diluted in 100 μl Opti-MEM I Reduced Serum Medium and incubated for 15 min at room temperature.

    Labeling:

    Article Title: siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro
    Article Snippet: A scrambled sequence with the same base composition as siRNA-69 was also designed as the negative control (siRNA-SCS) and shared no significant homology with the target gene. .. A BLOCK-iT Fluorescent Oligo (Invitrogen Life Technologies, Carlsbad, CA, USA) labeled with fluorescein isothiocyanate (FITC) was synthesized to test the transfection efficiency of the siRNAs. .. All the siRNAs were synthesized by Invitrogen.

    Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
    Article Snippet: Then, siRNAs were dissolved with Rnase free H2 O to the concentration of 20 μM. .. To determine the optimal transfection condition, various combinations of siRNA concentrations (25 nM, 50 nM and 100 nM FITC labeled nonsilencing siRNA, FITC-Oligo, Invitrogen) and transfection reagent volumes (0.5 μl, 1 μl, 1.5μl and 2 μl Lipofectamine 2000, Lipo 2000, Invitrogen) were examined. .. Before transfection, PFFs were cultured in 400 μl Opti-MEM (GIBCO), 0.5 μl, 1 μl, 1.5μl or 2 μl Lipo 2000 was added into Opti-MEM and incubated at room temperature for 5 min, 20 μM FITC-Oligo was diluted into 250 nM, 500 nM or 1000 nM with Opti-MEM, then, 100 μl FITC-Oligo-Lipo 2000 complexes was obtained through a mixture of 50 μl Opti-MEM with 250 nM, 500 nM or 1000 nM FITC-Oligo and 50 μl Opti-MEM with 0.5 μl, 1 μl, 1.5μl or 2 μl Lipo 2000, incubated at room temperature for 30 min and added into each 24-well culture plate with PFFs and 400 μl Opti-MEM.

    Synthesized:

    Article Title: siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro
    Article Snippet: A scrambled sequence with the same base composition as siRNA-69 was also designed as the negative control (siRNA-SCS) and shared no significant homology with the target gene. .. A BLOCK-iT Fluorescent Oligo (Invitrogen Life Technologies, Carlsbad, CA, USA) labeled with fluorescein isothiocyanate (FITC) was synthesized to test the transfection efficiency of the siRNAs. .. All the siRNAs were synthesized by Invitrogen.

    Over Expression:

    Article Title: miR-612 negatively regulates colorectal cancer growth and metastasis by targeting AKT2
    Article Snippet: Mutation and deletion of miR-612 binding sites in the AKT2 3′-UTR were achieved by site-directed mutagenesis using the QuikChangeH XL Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA, USA) according to the manufacturer's instructions. .. Cell transfection Oligonucleotides including miR-612 mimic and the miR-612 inhibitor anti-miR-612 were used (Thermo Scientific, Lafayette, CO, USA) for overexpression or inhibition of miR-612, respectively. .. For cell transfection assays, the synthetic oligonucleotides were transfected into cells using a Lipofectamine RNAiMAX Kit (Invitrogen) at about 50% confluence, according to the product manual.

    Inhibition:

    Article Title: miR-612 negatively regulates colorectal cancer growth and metastasis by targeting AKT2
    Article Snippet: Mutation and deletion of miR-612 binding sites in the AKT2 3′-UTR were achieved by site-directed mutagenesis using the QuikChangeH XL Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA, USA) according to the manufacturer's instructions. .. Cell transfection Oligonucleotides including miR-612 mimic and the miR-612 inhibitor anti-miR-612 were used (Thermo Scientific, Lafayette, CO, USA) for overexpression or inhibition of miR-612, respectively. .. For cell transfection assays, the synthetic oligonucleotides were transfected into cells using a Lipofectamine RNAiMAX Kit (Invitrogen) at about 50% confluence, according to the product manual.

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  • 97
    Thermo Fisher sirna
    Effect of HNF1A knockdown on AKT/mTOR signaling pathway was assessed by Western Blot. AsPC-1 and BxPC-3 cells were transfected with control <t>siRNA</t> and two different HNF1A siRNAs as indicated. 96h after transfection, cell lysates were immunoblotted with anti- HNF1A antibody, anti-phospho-AKT Ser473 antibody, anti-phospho-AKT Thr308 antibody, and anti-phospho-mTOR Ser2448 antibody. Membranes were stripped and re-probed with anti-AKT antibody, anti-mTOR antibody and anti-β-actin antibody. The fold differences in protein expression levels of cells transfected with HNF1A siRNA and control siRNA was presented as mean ± SD from three independent experiments.
    Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    99
    Thermo Fisher ocrl kd
    Autophagy flux is impaired due to PI(4,5)P 2 -mediated inhibition of MCOLN1 activity in <t>OCRL-depleted</t> cells. ( a ) Mock (CTRL), YU142670-treated and OCRL-KD (Sigma (S) or <t>Ambion</t> (A) siRNA pools) cells were incubated in HBSS for 3 hours and labeled with anti-LAMP1 (green) and anti-PI(4,5)P 2 (red) antibodies. White arrows indicate structures positive for both markers. ( b ) Quantification of PI(4,5)P 2 -LAMP1 colocalizing structures in control, YU142670-treated, OCRL-KD (-), OCRL-KD plus PIP5K1ß-KD, and OCRL-KD plus PIP5K1α-KD cells. Mean values ± s.d. n=200 cells per condition pooled from 4 independent experiments. ( c ) Quantification of LC3-positive structures in the cells described in b . Red line, mean values. ( d ) OCRL depletion impairs MCOLN1-dependent calcium release. Control or OCRL-KD cells loaded with the Ca 2+ -sensitive dye FuraRed were treated with 50 or 200 µM of the MCOLN1 agonist SF-51 47 . The ratio of fluorescence excited at 457 and 488 nm was measured and normalized against F0 (time-point before drug addition, black arrow). Plots correspond to the average of the response of n=20 cells from a single experiment, that was independently repeated 3 times. ( e ) Representative images and quantification of MCOLN1 and OCRL colocalization in cells transfected with wild type OCRL or the AP2-defective binding mutant (OCRL-F151S) along with MCOLN1-Myc. Right, colocalization of OCRL with MCOLN1. Mean values ± s.d. n=100 cells per condition pooled from 3 independent experiments. ( f ) MCOLN1-KD cells transfected with GFP-OCRL were treated with HBSS for 3 hours and stained for LAMP1 (red). Right, quantification of OCRL colocalization with LAMP1. Mean values ± s.d. n=100 cells per condition pooled from 3 independent experiments. ( g ) Cell lysates from HK-2 cells were immunoprecipitated using an anti-MCOLN1 antibody and analyzed by Western blot (WB) with anti-MCOLN1 or anti-OCRL antibodies. The results are representative of five independent experiments. An unprocessed scan of the blot is shown in Supplementary Fig. 9 . Parallel samples were analyzed by LC-MS/MS and peptides significantly matching human OCRL (Q01968-2) were found in anti-MCOLN1 immunoprecipitates but not in control-IgG immunoprecipitates. p-values calculated by One-way ANOVA with Tukey's post hoc test. Statistic source data can be found in Supplementary Table 2 . Scale bars, 10 μm.
    Ocrl Kd, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher human rnf126
    <t>RNF126</t> ubiquitylates Ku80 and Ku70 in an IR-dependent manner. (A) HEK293T cells transfected with expression vectors for the indicated proteins and incubated with the proteasome inhibitor MG132 (10 μM) for 5 h were lysed and subjected to Ni 2+ -agarose
    Human Rnf126, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of HNF1A knockdown on AKT/mTOR signaling pathway was assessed by Western Blot. AsPC-1 and BxPC-3 cells were transfected with control siRNA and two different HNF1A siRNAs as indicated. 96h after transfection, cell lysates were immunoblotted with anti- HNF1A antibody, anti-phospho-AKT Ser473 antibody, anti-phospho-AKT Thr308 antibody, and anti-phospho-mTOR Ser2448 antibody. Membranes were stripped and re-probed with anti-AKT antibody, anti-mTOR antibody and anti-β-actin antibody. The fold differences in protein expression levels of cells transfected with HNF1A siRNA and control siRNA was presented as mean ± SD from three independent experiments.

    Journal: PLoS ONE

    Article Title: Hepatocyte Nuclear Factor 1A (HNF1A) as a Possible Tumor Suppressor in Pancreatic Cancer

    doi: 10.1371/journal.pone.0121082

    Figure Lengend Snippet: Effect of HNF1A knockdown on AKT/mTOR signaling pathway was assessed by Western Blot. AsPC-1 and BxPC-3 cells were transfected with control siRNA and two different HNF1A siRNAs as indicated. 96h after transfection, cell lysates were immunoblotted with anti- HNF1A antibody, anti-phospho-AKT Ser473 antibody, anti-phospho-AKT Thr308 antibody, and anti-phospho-mTOR Ser2448 antibody. Membranes were stripped and re-probed with anti-AKT antibody, anti-mTOR antibody and anti-β-actin antibody. The fold differences in protein expression levels of cells transfected with HNF1A siRNA and control siRNA was presented as mean ± SD from three independent experiments.

    Article Snippet: siRNA blocking Small interfering RNA (siRNA) transfections were performed using transfection reagent according to the manufacturer’s protocol (Life Technologies, Grand Island, NY).

    Techniques: Western Blot, Transfection, Expressing

    HNF1A knockdown by specific siRNAs in AsPC-1 (A) and BxPC-3 (B) cells. Cells were transfected independently with three different sequences of siRNA directed against HNF1A (siRNA1, siRNA2, siRNA3), or with a control siRNA (Control). Inhibition efficiencies were assessed by qPCR for relative levels of HNF1A mRNA at 24 h to 96 h following the transfections. Western Blot analyses of AsPC-1 and BxPC-3 cells transfected with anti-HNF1A siRNAs. Data are shown for 96 h following transfections. Expression of β-actin was used as an internal control.

    Journal: PLoS ONE

    Article Title: Hepatocyte Nuclear Factor 1A (HNF1A) as a Possible Tumor Suppressor in Pancreatic Cancer

    doi: 10.1371/journal.pone.0121082

    Figure Lengend Snippet: HNF1A knockdown by specific siRNAs in AsPC-1 (A) and BxPC-3 (B) cells. Cells were transfected independently with three different sequences of siRNA directed against HNF1A (siRNA1, siRNA2, siRNA3), or with a control siRNA (Control). Inhibition efficiencies were assessed by qPCR for relative levels of HNF1A mRNA at 24 h to 96 h following the transfections. Western Blot analyses of AsPC-1 and BxPC-3 cells transfected with anti-HNF1A siRNAs. Data are shown for 96 h following transfections. Expression of β-actin was used as an internal control.

    Article Snippet: siRNA blocking Small interfering RNA (siRNA) transfections were performed using transfection reagent according to the manufacturer’s protocol (Life Technologies, Grand Island, NY).

    Techniques: Transfection, Inhibition, Real-time Polymerase Chain Reaction, Western Blot, Expressing

    Effect of HNF1A knockdown on cell proliferation and cell cycle. Pancreatic cancer cell lines were transfected with one of the two HNF1A siRNAs or a control siRNA. The proliferation rate of the siRNA-transfected cells was assessed by CellTiter-Glo assay on AsPC-1 (A) and BxPC-3 (B) cells 12 h to 72h after transfection. Cell cycle distribution was determined by Fluorescence-activated cell sorting analysis in AsPC-1(C) and BxPC-3 (D) cells. Cells were stained with PI, and analyzed for DNA content using flow cytometry 72h following transfections. The populations of cells in G1, S, and G2 phases are characterized by different intensities of PI-A fluorescence and are indicated. Data are shown relative to the control. *P

    Journal: PLoS ONE

    Article Title: Hepatocyte Nuclear Factor 1A (HNF1A) as a Possible Tumor Suppressor in Pancreatic Cancer

    doi: 10.1371/journal.pone.0121082

    Figure Lengend Snippet: Effect of HNF1A knockdown on cell proliferation and cell cycle. Pancreatic cancer cell lines were transfected with one of the two HNF1A siRNAs or a control siRNA. The proliferation rate of the siRNA-transfected cells was assessed by CellTiter-Glo assay on AsPC-1 (A) and BxPC-3 (B) cells 12 h to 72h after transfection. Cell cycle distribution was determined by Fluorescence-activated cell sorting analysis in AsPC-1(C) and BxPC-3 (D) cells. Cells were stained with PI, and analyzed for DNA content using flow cytometry 72h following transfections. The populations of cells in G1, S, and G2 phases are characterized by different intensities of PI-A fluorescence and are indicated. Data are shown relative to the control. *P

    Article Snippet: siRNA blocking Small interfering RNA (siRNA) transfections were performed using transfection reagent according to the manufacturer’s protocol (Life Technologies, Grand Island, NY).

    Techniques: Transfection, Glo Assay, Fluorescence, FACS, Staining, Flow Cytometry, Cytometry

    Autophagy flux is impaired due to PI(4,5)P 2 -mediated inhibition of MCOLN1 activity in OCRL-depleted cells. ( a ) Mock (CTRL), YU142670-treated and OCRL-KD (Sigma (S) or Ambion (A) siRNA pools) cells were incubated in HBSS for 3 hours and labeled with anti-LAMP1 (green) and anti-PI(4,5)P 2 (red) antibodies. White arrows indicate structures positive for both markers. ( b ) Quantification of PI(4,5)P 2 -LAMP1 colocalizing structures in control, YU142670-treated, OCRL-KD (-), OCRL-KD plus PIP5K1ß-KD, and OCRL-KD plus PIP5K1α-KD cells. Mean values ± s.d. n=200 cells per condition pooled from 4 independent experiments. ( c ) Quantification of LC3-positive structures in the cells described in b . Red line, mean values. ( d ) OCRL depletion impairs MCOLN1-dependent calcium release. Control or OCRL-KD cells loaded with the Ca 2+ -sensitive dye FuraRed were treated with 50 or 200 µM of the MCOLN1 agonist SF-51 47 . The ratio of fluorescence excited at 457 and 488 nm was measured and normalized against F0 (time-point before drug addition, black arrow). Plots correspond to the average of the response of n=20 cells from a single experiment, that was independently repeated 3 times. ( e ) Representative images and quantification of MCOLN1 and OCRL colocalization in cells transfected with wild type OCRL or the AP2-defective binding mutant (OCRL-F151S) along with MCOLN1-Myc. Right, colocalization of OCRL with MCOLN1. Mean values ± s.d. n=100 cells per condition pooled from 3 independent experiments. ( f ) MCOLN1-KD cells transfected with GFP-OCRL were treated with HBSS for 3 hours and stained for LAMP1 (red). Right, quantification of OCRL colocalization with LAMP1. Mean values ± s.d. n=100 cells per condition pooled from 3 independent experiments. ( g ) Cell lysates from HK-2 cells were immunoprecipitated using an anti-MCOLN1 antibody and analyzed by Western blot (WB) with anti-MCOLN1 or anti-OCRL antibodies. The results are representative of five independent experiments. An unprocessed scan of the blot is shown in Supplementary Fig. 9 . Parallel samples were analyzed by LC-MS/MS and peptides significantly matching human OCRL (Q01968-2) were found in anti-MCOLN1 immunoprecipitates but not in control-IgG immunoprecipitates. p-values calculated by One-way ANOVA with Tukey's post hoc test. Statistic source data can be found in Supplementary Table 2 . Scale bars, 10 μm.

    Journal: Nature cell biology

    Article Title: Autophagosome-lysosome fusion triggers a lysosomal response mediated by TLR9 and controlled by OCRL

    doi: 10.1038/ncb3386

    Figure Lengend Snippet: Autophagy flux is impaired due to PI(4,5)P 2 -mediated inhibition of MCOLN1 activity in OCRL-depleted cells. ( a ) Mock (CTRL), YU142670-treated and OCRL-KD (Sigma (S) or Ambion (A) siRNA pools) cells were incubated in HBSS for 3 hours and labeled with anti-LAMP1 (green) and anti-PI(4,5)P 2 (red) antibodies. White arrows indicate structures positive for both markers. ( b ) Quantification of PI(4,5)P 2 -LAMP1 colocalizing structures in control, YU142670-treated, OCRL-KD (-), OCRL-KD plus PIP5K1ß-KD, and OCRL-KD plus PIP5K1α-KD cells. Mean values ± s.d. n=200 cells per condition pooled from 4 independent experiments. ( c ) Quantification of LC3-positive structures in the cells described in b . Red line, mean values. ( d ) OCRL depletion impairs MCOLN1-dependent calcium release. Control or OCRL-KD cells loaded with the Ca 2+ -sensitive dye FuraRed were treated with 50 or 200 µM of the MCOLN1 agonist SF-51 47 . The ratio of fluorescence excited at 457 and 488 nm was measured and normalized against F0 (time-point before drug addition, black arrow). Plots correspond to the average of the response of n=20 cells from a single experiment, that was independently repeated 3 times. ( e ) Representative images and quantification of MCOLN1 and OCRL colocalization in cells transfected with wild type OCRL or the AP2-defective binding mutant (OCRL-F151S) along with MCOLN1-Myc. Right, colocalization of OCRL with MCOLN1. Mean values ± s.d. n=100 cells per condition pooled from 3 independent experiments. ( f ) MCOLN1-KD cells transfected with GFP-OCRL were treated with HBSS for 3 hours and stained for LAMP1 (red). Right, quantification of OCRL colocalization with LAMP1. Mean values ± s.d. n=100 cells per condition pooled from 3 independent experiments. ( g ) Cell lysates from HK-2 cells were immunoprecipitated using an anti-MCOLN1 antibody and analyzed by Western blot (WB) with anti-MCOLN1 or anti-OCRL antibodies. The results are representative of five independent experiments. An unprocessed scan of the blot is shown in Supplementary Fig. 9 . Parallel samples were analyzed by LC-MS/MS and peptides significantly matching human OCRL (Q01968-2) were found in anti-MCOLN1 immunoprecipitates but not in control-IgG immunoprecipitates. p-values calculated by One-way ANOVA with Tukey's post hoc test. Statistic source data can be found in Supplementary Table 2 . Scale bars, 10 μm.

    Article Snippet: Supplementary Figure 2 OCRL depletion induces upregulation of lysosomal genes and TFEB nuclear translocation. ( a ) Fold change expression of the indicated lysosomal genes (selected amongst those most upregulated in the microarray) in OCRL-KD (Ambion siRNAs pool) cells relative to non-targeting siRNA-treated cells (CTRL) as assessed by quantitative RT-PCR carried out in triplicate and the data represent mean values ± s.d. from n=6 independent experiments. p values calculated using Student’s t test (unpaired). ( b ) CTRL, OCRL-KD (treated either with Sigma (S) or Ambion (A) pools) and YU142670-treated HK-2 cells were stained with an anti-TFEB antibody (green).

    Techniques: Inhibition, Activity Assay, Incubation, Labeling, Fluorescence, Transfection, Binding Assay, Mutagenesis, Staining, Immunoprecipitation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Autophagosome-lysosome fusion induces an AP2 and clathrin-dependent recruitment of OCRL to lysosomes. ( a ) Fusion of autophagosomes with lysosomes recruits OCRL to lysosomes. HK-2 cells were incubated in growth medium, or in HBSS for 3 hours (-), or in HBSS for 3 hours after impairment of autophagosome-lysosome fusion by siRNA-mediated KD of the HOPS component VPS16 12 or of the autophagosomal SNARE STX17 14 . Cells were stained for OCRL and LAMP1 (a lysosomal marker). The lower panels are enlargements of the boxed areas and fluorescence intensity profiles in the green and red channels of the regions underneath the white lines. Scale bars, 10 µm. ( b ) Quantification of OCRL association with lysosomes under conditions described in a , or after the addition of vinblastine (20 μM) that also inhibits autophagosome-lysosome fusion 13 . The percentage of LAMP1 structures that are positive for OCRL, the percentage of LC3 structures that are positive for LAMP1 (as a measure of the arrival of autophagosomal cargo to lysosomes), and the average number of LC3 structures per cell are reported. Values are means ± s.d. of n=450 cells pooled from 3 independent experiments. NS (not significant). ( c ) Representative images and quantification of the colocalization of wt OCRL, the AP2 (OCRL-F151S), clathrin(OCRL-I74N) or AP2-clathrin triple (OCRL-X3) binding mutants, and INPP5B with LAMP1. Insets, enlargements of the boxed areas; lower panels, fluorescence intensity profiles in the green and red channels of the regions underneath the white lines. Scale bars, 10 µm. Data represent the percentage of total LAMP1 structures positive for each OCRL form or for INPP5B. Means ± s.d. n=200 cells pooled from 3 independent experiments; n=100 OCRL-X3 transfected cells pooled from 3 independent experiments. p-values calculated by One-way ANOVA with Tukey's post hoc test.

    Journal: Nature cell biology

    Article Title: Autophagosome-lysosome fusion triggers a lysosomal response mediated by TLR9 and controlled by OCRL

    doi: 10.1038/ncb3386

    Figure Lengend Snippet: Autophagosome-lysosome fusion induces an AP2 and clathrin-dependent recruitment of OCRL to lysosomes. ( a ) Fusion of autophagosomes with lysosomes recruits OCRL to lysosomes. HK-2 cells were incubated in growth medium, or in HBSS for 3 hours (-), or in HBSS for 3 hours after impairment of autophagosome-lysosome fusion by siRNA-mediated KD of the HOPS component VPS16 12 or of the autophagosomal SNARE STX17 14 . Cells were stained for OCRL and LAMP1 (a lysosomal marker). The lower panels are enlargements of the boxed areas and fluorescence intensity profiles in the green and red channels of the regions underneath the white lines. Scale bars, 10 µm. ( b ) Quantification of OCRL association with lysosomes under conditions described in a , or after the addition of vinblastine (20 μM) that also inhibits autophagosome-lysosome fusion 13 . The percentage of LAMP1 structures that are positive for OCRL, the percentage of LC3 structures that are positive for LAMP1 (as a measure of the arrival of autophagosomal cargo to lysosomes), and the average number of LC3 structures per cell are reported. Values are means ± s.d. of n=450 cells pooled from 3 independent experiments. NS (not significant). ( c ) Representative images and quantification of the colocalization of wt OCRL, the AP2 (OCRL-F151S), clathrin(OCRL-I74N) or AP2-clathrin triple (OCRL-X3) binding mutants, and INPP5B with LAMP1. Insets, enlargements of the boxed areas; lower panels, fluorescence intensity profiles in the green and red channels of the regions underneath the white lines. Scale bars, 10 µm. Data represent the percentage of total LAMP1 structures positive for each OCRL form or for INPP5B. Means ± s.d. n=200 cells pooled from 3 independent experiments; n=100 OCRL-X3 transfected cells pooled from 3 independent experiments. p-values calculated by One-way ANOVA with Tukey's post hoc test.

    Article Snippet: Supplementary Figure 2 OCRL depletion induces upregulation of lysosomal genes and TFEB nuclear translocation. ( a ) Fold change expression of the indicated lysosomal genes (selected amongst those most upregulated in the microarray) in OCRL-KD (Ambion siRNAs pool) cells relative to non-targeting siRNA-treated cells (CTRL) as assessed by quantitative RT-PCR carried out in triplicate and the data represent mean values ± s.d. from n=6 independent experiments. p values calculated using Student’s t test (unpaired). ( b ) CTRL, OCRL-KD (treated either with Sigma (S) or Ambion (A) pools) and YU142670-treated HK-2 cells were stained with an anti-TFEB antibody (green).

    Techniques: Incubation, Staining, Marker, Fluorescence, Binding Assay, Transfection

    OCRL depletion/mutation induces upregulation of lysosomal genes and morphological changes in lysosomes. ( a ) Volcano plot of OCRL-KD gene expression data. Horizontal black line: -log 10 of FDR (False Discovery Rate, significance threshold 0.05); vertical black lines: log 2 fold change (1.3-fold threshold). 910 genes up-regulated above (red) and 630 genes down-regulated below (green) the threshold are shown. Black dots indicate 24 upregulated (35 probe sets) out of the 194 lysosomal genes annotated in the Lysoplex list 50 . ( b ) Gene set enrichment analysis of the data in a . Subcellular compartments, except lysosomes, were defined according to the Human Protein Atlas. Lysosomal genes were defined according to Lysoplex 50 and excluded from the other categories. p-values calculated as described in Methods . (c-e) Lysosomal enlargement in OCRL-depleted HK-2 cells, in PTCs of Lowe syndrome patients, and in the proximal pronephric tubule of ocrl-/- zebrafish embryos. ( c ) Representative images of Control and OCRL-KD cells immunostained with an anti-LAMP1 antibody (upper panels, scale bars, 10 µm) or immunoelectron-microscopy with an anti-LAMP1 antibody (lower panels, scale bars, 250 nm). ( d ) PTCs obtained from the urine of healthy control subjects (CTRL) and Lowe Syndrome patients were processed as in a . Black arrows indicate LAMP1-positive structures. ( e ) Confocal transverse sections of zebrafish proximal pronephric tubules from 72 hpf GFP-LAMP1-expressing wild-type (wt) and ocrl-/- mutant embryos 9 labelled with an anti-GFP antibody. White dashed lines indicate the outline of pronephric tubules (upper panels). Scale bars, 5 µm. Block face scanning electron microscopy images of transverse sections through proximal pronephric tubules from 72 hpf wild-type (wt) or ocrl-/- embryos (lower panels). Scale bar, 5 µm. Graphs show the morphometric analysis of LAMP1-positive structures in c [n=81 (CTRL) and n=126 (OCRL KD) structures pooled from 3 independent experiments] and d [n=102 (CTRL), n=84 (Lowe) structures pooled from 3 independent experiments]. In e the external circumference of each lysosomal compartment (electron dense oval or spherical membrane-enclosed organelle) was manually traced in each section and the average calculated. n=30 sections (wt); n=30 sections (ocrl-/-) (10 sections analyzed for three kidneys per category). Data are presented as means ± s.d. ***p

    Journal: Nature cell biology

    Article Title: Autophagosome-lysosome fusion triggers a lysosomal response mediated by TLR9 and controlled by OCRL

    doi: 10.1038/ncb3386

    Figure Lengend Snippet: OCRL depletion/mutation induces upregulation of lysosomal genes and morphological changes in lysosomes. ( a ) Volcano plot of OCRL-KD gene expression data. Horizontal black line: -log 10 of FDR (False Discovery Rate, significance threshold 0.05); vertical black lines: log 2 fold change (1.3-fold threshold). 910 genes up-regulated above (red) and 630 genes down-regulated below (green) the threshold are shown. Black dots indicate 24 upregulated (35 probe sets) out of the 194 lysosomal genes annotated in the Lysoplex list 50 . ( b ) Gene set enrichment analysis of the data in a . Subcellular compartments, except lysosomes, were defined according to the Human Protein Atlas. Lysosomal genes were defined according to Lysoplex 50 and excluded from the other categories. p-values calculated as described in Methods . (c-e) Lysosomal enlargement in OCRL-depleted HK-2 cells, in PTCs of Lowe syndrome patients, and in the proximal pronephric tubule of ocrl-/- zebrafish embryos. ( c ) Representative images of Control and OCRL-KD cells immunostained with an anti-LAMP1 antibody (upper panels, scale bars, 10 µm) or immunoelectron-microscopy with an anti-LAMP1 antibody (lower panels, scale bars, 250 nm). ( d ) PTCs obtained from the urine of healthy control subjects (CTRL) and Lowe Syndrome patients were processed as in a . Black arrows indicate LAMP1-positive structures. ( e ) Confocal transverse sections of zebrafish proximal pronephric tubules from 72 hpf GFP-LAMP1-expressing wild-type (wt) and ocrl-/- mutant embryos 9 labelled with an anti-GFP antibody. White dashed lines indicate the outline of pronephric tubules (upper panels). Scale bars, 5 µm. Block face scanning electron microscopy images of transverse sections through proximal pronephric tubules from 72 hpf wild-type (wt) or ocrl-/- embryos (lower panels). Scale bar, 5 µm. Graphs show the morphometric analysis of LAMP1-positive structures in c [n=81 (CTRL) and n=126 (OCRL KD) structures pooled from 3 independent experiments] and d [n=102 (CTRL), n=84 (Lowe) structures pooled from 3 independent experiments]. In e the external circumference of each lysosomal compartment (electron dense oval or spherical membrane-enclosed organelle) was manually traced in each section and the average calculated. n=30 sections (wt); n=30 sections (ocrl-/-) (10 sections analyzed for three kidneys per category). Data are presented as means ± s.d. ***p

    Article Snippet: Supplementary Figure 2 OCRL depletion induces upregulation of lysosomal genes and TFEB nuclear translocation. ( a ) Fold change expression of the indicated lysosomal genes (selected amongst those most upregulated in the microarray) in OCRL-KD (Ambion siRNAs pool) cells relative to non-targeting siRNA-treated cells (CTRL) as assessed by quantitative RT-PCR carried out in triplicate and the data represent mean values ± s.d. from n=6 independent experiments. p values calculated using Student’s t test (unpaired). ( b ) CTRL, OCRL-KD (treated either with Sigma (S) or Ambion (A) pools) and YU142670-treated HK-2 cells were stained with an anti-TFEB antibody (green).

    Techniques: Mutagenesis, Expressing, Immuno-Electron Microscopy, Blocking Assay, Electron Microscopy

    RNF126 ubiquitylates Ku80 and Ku70 in an IR-dependent manner. (A) HEK293T cells transfected with expression vectors for the indicated proteins and incubated with the proteasome inhibitor MG132 (10 μM) for 5 h were lysed and subjected to Ni 2+ -agarose

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair

    doi: 10.1128/MCB.00347-16

    Figure Lengend Snippet: RNF126 ubiquitylates Ku80 and Ku70 in an IR-dependent manner. (A) HEK293T cells transfected with expression vectors for the indicated proteins and incubated with the proteasome inhibitor MG132 (10 μM) for 5 h were lysed and subjected to Ni 2+ -agarose

    Article Snippet: Small interfering RNAs were described previously for Ku80 , were designed with the use of BLOCK-iT RNAi designer for human RNF126 (Stealth siRNA; ), or were obtained from Life Technologies for a negative-control duplex (medium GC duplex, no. 12935-300).

    Techniques: Transfection, Expressing, Incubation

    RNF126 promotes IR-dependent degradation of Ku80 and Ku70 associated with chromatin. (A) U2OS cells were exposed (or left unexposed) to IR (2 Gy), incubated with cycloheximide (50 μg/ml) in the absence or presence of 10 μM MG132 (MG) for

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair

    doi: 10.1128/MCB.00347-16

    Figure Lengend Snippet: RNF126 promotes IR-dependent degradation of Ku80 and Ku70 associated with chromatin. (A) U2OS cells were exposed (or left unexposed) to IR (2 Gy), incubated with cycloheximide (50 μg/ml) in the absence or presence of 10 μM MG132 (MG) for

    Article Snippet: Small interfering RNAs were described previously for Ku80 , were designed with the use of BLOCK-iT RNAi designer for human RNF126 (Stealth siRNA; ), or were obtained from Life Technologies for a negative-control duplex (medium GC duplex, no. 12935-300).

    Techniques: Incubation

    Ubiquitylation of Ku80 by RNF126 is required for DSB repair by NHEJ. (A) Schematic representation of the KR19 mutant of human Ku80, in which predicted RNF126 ubiquitylation sites are replaced with arginine. (B) In vitro ubiquitylation assay for WT and

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair

    doi: 10.1128/MCB.00347-16

    Figure Lengend Snippet: Ubiquitylation of Ku80 by RNF126 is required for DSB repair by NHEJ. (A) Schematic representation of the KR19 mutant of human Ku80, in which predicted RNF126 ubiquitylation sites are replaced with arginine. (B) In vitro ubiquitylation assay for WT and

    Article Snippet: Small interfering RNAs were described previously for Ku80 , were designed with the use of BLOCK-iT RNAi designer for human RNF126 (Stealth siRNA; ), or were obtained from Life Technologies for a negative-control duplex (medium GC duplex, no. 12935-300).

    Techniques: Non-Homologous End Joining, Mutagenesis, In Vitro, Ubiquitin Assay

    RNF126 accumulates at DSB sites and regulates dissociation of Ku70/80 from sites of DNA damage. (A) U2OS cells expressing EGFP-tagged RNF126 or Ku80 were monitored for EGFP fluorescence before and 10 min after laser microirradiation. Arrowheads indicate

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair

    doi: 10.1128/MCB.00347-16

    Figure Lengend Snippet: RNF126 accumulates at DSB sites and regulates dissociation of Ku70/80 from sites of DNA damage. (A) U2OS cells expressing EGFP-tagged RNF126 or Ku80 were monitored for EGFP fluorescence before and 10 min after laser microirradiation. Arrowheads indicate

    Article Snippet: Small interfering RNAs were described previously for Ku80 , were designed with the use of BLOCK-iT RNAi designer for human RNF126 (Stealth siRNA; ), or were obtained from Life Technologies for a negative-control duplex (medium GC duplex, no. 12935-300).

    Techniques: Expressing, Fluorescence

    RNF126 interacts with the Ku70-Ku80 heterodimer. (A) Flp-In T-REx 293-RNF126 cells were exposed (or left unexposed) to Tet or gamma radiation (IR; 2 Gy), lysed, and subjected to immunoprecipitation (IP) with antibodies to FLAG. The resulting precipitates,

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair

    doi: 10.1128/MCB.00347-16

    Figure Lengend Snippet: RNF126 interacts with the Ku70-Ku80 heterodimer. (A) Flp-In T-REx 293-RNF126 cells were exposed (or left unexposed) to Tet or gamma radiation (IR; 2 Gy), lysed, and subjected to immunoprecipitation (IP) with antibodies to FLAG. The resulting precipitates,

    Article Snippet: Small interfering RNAs were described previously for Ku80 , were designed with the use of BLOCK-iT RNAi designer for human RNF126 (Stealth siRNA; ), or were obtained from Life Technologies for a negative-control duplex (medium GC duplex, no. 12935-300).

    Techniques: Immunoprecipitation

    Regulation of Ku80 by RNF126. Model for the role of RNF126 in the ubiquitylation of Ku and its subsequent removal from DSB sites and degradation in the NHEJ pathway of DNA repair.

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair

    doi: 10.1128/MCB.00347-16

    Figure Lengend Snippet: Regulation of Ku80 by RNF126. Model for the role of RNF126 in the ubiquitylation of Ku and its subsequent removal from DSB sites and degradation in the NHEJ pathway of DNA repair.

    Article Snippet: Small interfering RNAs were described previously for Ku80 , were designed with the use of BLOCK-iT RNAi designer for human RNF126 (Stealth siRNA; ), or were obtained from Life Technologies for a negative-control duplex (medium GC duplex, no. 12935-300).

    Techniques: Non-Homologous End Joining

    RNF126 is required for completion of NHEJ. (A) U2OS cells were transfected with RNF126 or control siRNAs, treated (or not treated [NT]) with bleomycin, incubated for the indicated times, and then subjected to a single-cell neutral gel electrophoresis

    Journal: Molecular and Cellular Biology

    Article Title: Ubiquitylation of Ku80 by RNF126 Promotes Completion of Nonhomologous End Joining-Mediated DNA Repair

    doi: 10.1128/MCB.00347-16

    Figure Lengend Snippet: RNF126 is required for completion of NHEJ. (A) U2OS cells were transfected with RNF126 or control siRNAs, treated (or not treated [NT]) with bleomycin, incubated for the indicated times, and then subjected to a single-cell neutral gel electrophoresis

    Article Snippet: Small interfering RNAs were described previously for Ku80 , were designed with the use of BLOCK-iT RNAi designer for human RNF126 (Stealth siRNA; ), or were obtained from Life Technologies for a negative-control duplex (medium GC duplex, no. 12935-300).

    Techniques: Non-Homologous End Joining, Transfection, Incubation, Nucleic Acid Electrophoresis