Journal: Developmental biology

Article Title: Dgcr8 controls neural crest cells survival in cardiovascular development.

doi: 10.1016/j.ydbio.2011.11.008

Figure Lengend Snippet: Fig. 1. Severe cardiovascular malformations observed in Dgcr8 mutants. Sagittal aortic arch sections of E10.5 Wnt1-cre;Dgcr8loxp/+;R26R-YFP controls (A, n=3) and Wnt1-cre; Dgcr8loxp/loxp;R26R-YFP mutants (B, n=4) stained for YFP (green) and DGCR8 (red). Magnified view in insets. (C–E) Gross thorax anatomy of E17.5 controls (C, n=10) and Wnt1-cre;Dgcr8loxp/loxp mutants (D–E, n=11) and respective schemes of the great vessels (C′–E′). All Wnt1-cre;Dgcr8loxp/loxp displayed persistent truncus arteriosus (PTA; D′,E′ n=11/11), while additional abnormalities included interrupted aortic arch type B (IAA; E′ n=3/11), aberrant origin of the right subclavian artery (Ab-RSA; D′ n=3/11) and cer- vical aortic arch (Cx-AA; D′ n=2/11). RSA, right subclavian artery; RCC, right common carotid; IA, inominate artery; LSA, left subclavian artery; LCC, left common carotid; AA, aortic arch; Ao, aorta; DA, ductus arteriosus. Anterior (top) view of E18.5 OFT, divided into pulmonary artery and aorta in healthy controls (F) and displays persistent truncus arteriosus in Wnt1-cre;Dgcr8loxp/loxp (G). H&E staining of representative E18.5 frontal heart sections from controls (H, n=3) and Wnt1-cre;Dgcr8loxp/loxp mutants (I, n=3), the latter manifests with persistent truncus arteriosus (PTA) and ventricular septal defect (VSD). Ao, aorta; PA, pulmonary artery; RV, right ventricle; LV, left ventricle. Frontal view of E18.5 control thymus (J, n=4) and Wnt1-cre;Dgcr8loxp/loxp thymus that is malformed and ectopically positioned, in a lateral situs in the thorax (K, n=3). LT, left thymic lobe; RT, right thymic lobe; Tr, trachea.

Article Snippet: Primary antibodies in CAS-Block were incubated overnight at 4 °C: DGCR8 (10996-1-AP, 1:100 dilution, Proteintech), GFP (ab6658, 1:300, Abcam), Smooth muscle α-actin (A 2547, 1:400, DAKO), cleaved Caspase-3 (9661, 1:300, Cell Signaling Technology).

Techniques: Staining, Control

Journal: Developmental biology

Article Title: Dgcr8 controls neural crest cells survival in cardiovascular development.

doi: 10.1016/j.ydbio.2011.11.008

Figure Lengend Snippet: Fig. 2. Normal migration but reduced number of neural crest-descendants in Dgcr8 mutant OFT. India ink-injected E10.5 pharyngeal arch arteries. Lateral view of control (A, n=24) and Wnt1-cre;Dgcr8loxp/loxp mutant (B, n=8). 3, 3rd PAA; 4, 4th PAA; 6, 6th PAA. Whole mount in situ hybridization study of Ap2-alpha (C,D, n=5, per genotype), and Crabp1 (E,F, n=5 per genotype) on E10.5 Wnt1-cre;Dgcr8loxp/loxp embryos (D,F) and control littermates (C,E). Arrows indicate the migratory streams of NCCs entering the pharyngeal arches. Whole-mount beta-gal staining of E10.5 embryos, expressing a R26R-LacZ lineage tracing reporter in control (G) and Wnt1-cre;Dgcr8loxp/loxp (H) embryos (n=4, per genotype). Higher magnification reveals seemingly-normal cNCCs migration into the pharyngeal arches and OFT of controls (I) and Wnt1-cre;Dgcr8loxp/loxp (J). PA1, 1st pharyngeal arch. A black arrowhead denotes the distal-most LacZ-positive cells in the OFT. Sagittal sections of E10.5 embryos confirm the presence of NC-derived, LacZ-positive cells, in the 1st pha- ryngeal arch and the developing cardiac OFT, of control littermates (K) and Wnt1-cre;Dgcr8loxp/loxp mutants (L). Whole-mount beta-gal staining of E12.5 embryonic hearts expres- sing R26R-LacZ, reveals reduced cNCC numbers at the OFT of Wnt1-cre;Dgcr8loxp/loxp mutants (N, n=4, denoted by a black arrowhead), relative to controls (M, n=3). Ao, aorta; PA, pulmonary artery; PTA, persistent truncus arteriosus.

Article Snippet: Primary antibodies in CAS-Block were incubated overnight at 4 °C: DGCR8 (10996-1-AP, 1:100 dilution, Proteintech), GFP (ab6658, 1:300, Abcam), Smooth muscle α-actin (A 2547, 1:400, DAKO), cleaved Caspase-3 (9661, 1:300, Cell Signaling Technology).

Techniques: Migration, Mutagenesis, Injection, Control, In Situ Hybridization, Staining, Expressing, Derivative Assay

Journal: Developmental biology

Article Title: Dgcr8 controls neural crest cells survival in cardiovascular development.

doi: 10.1016/j.ydbio.2011.11.008

Figure Lengend Snippet: Fig. 3. Dgcr8 mutant cNCC descendants exhibit normal proliferation and smooth muscle differentiation. Immunofluorescent analysis of E10.5 sagittal sections reveals co-localization of α-smooth muscle actin (SMA-α, red) and cNCC genetic fate tracer R26R-YFP (Green) in the OFT of control (A, n=3) and Wnt1-cre;Dgcr8loxp/loxp mutant (B, n=3). Yellow, merged green and red signals. Representative cells at the OFT, positive for SMA-α and for R26R-YFP, are denoted by white arrowheads in A and B, respectively. Transverse sections of E10.5 embryos at the level of the OFT reveals comparable levels of BrdU incorporation in E10.5 Wnt1-cre;Dgcr8loxp/loxp (D) relative to controls (C). Red, BrdU; Green, YFP; Blue, DAPI. (E) Quantification of the percentage of BrdU-positive cells out of the YFP-positive (cNCCs) in mutants (red) and controls (blue, n=4 per genotype). n.s. not statistically significant.

Article Snippet: Primary antibodies in CAS-Block were incubated overnight at 4 °C: DGCR8 (10996-1-AP, 1:100 dilution, Proteintech), GFP (ab6658, 1:300, Abcam), Smooth muscle α-actin (A 2547, 1:400, DAKO), cleaved Caspase-3 (9661, 1:300, Cell Signaling Technology).

Techniques: Mutagenesis, Control, BrdU Incorporation Assay

Journal: Developmental biology

Article Title: Dgcr8 controls neural crest cells survival in cardiovascular development.

doi: 10.1016/j.ydbio.2011.11.008

Figure Lengend Snippet: Fig. 4. Dgcr8 mutant cNCC descendants exhibit enhanced cell death and attenuated ERK signaling. Cleaved caspase 3 (cCasp-3) immunofluorescent analysis of E10.5 sagittal pha- ryngeal arch (A,C) or outflow tract (OFT, B,D) sections. PA1-6 denote the 1st to the 6th pharyngeal arches, respectively; (n=3 per genotype). (E) Quantification of cleaved caspase 3 (cCasp-3) immunoreactive cells reveals enhanced apoptosis in the pharyngeal arch region of Wnt1-cre;Dgcr8loxp/loxp mutant (red) relative to control (blue), but not in the OFT per-se. * pb0.05. n.s. not statistically significant. Immunofluorescent analysis of E10.5 sagittal pharyngeal arch sections reveals significant downregulation of phosphorylated Erk1/2 in Wnt1-cre;Dgcr8loxp/loxp 1st and 2nd pharyngeal arches (I–K, J,K are enlargements of insets in I), relative to controls (F–H, G,H are enlarged micrographs of insets in F. n=3, per genotype).

Article Snippet: Primary antibodies in CAS-Block were incubated overnight at 4 °C: DGCR8 (10996-1-AP, 1:100 dilution, Proteintech), GFP (ab6658, 1:300, Abcam), Smooth muscle α-actin (A 2547, 1:400, DAKO), cleaved Caspase-3 (9661, 1:300, Cell Signaling Technology).

Techniques: Mutagenesis, Control

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Simtron biogenesis involves Drosha but not DGCR8. Knockdown of DGCR8 in HeLa cells using siRNA was quantitated by ( A ) RT–PCR analysis of DGCR8 mRNA and ( B ) western blot analysis of DGCR8 protein expression. The percentage of knockdown of DGCR8 was quantitated for DGCR8 mRNA using the equation 100 − [(( DGCR8 knockdown / GAPDH )/( DGCR8 control / GAPDH )) × 100], n = 5 and for DGCR8 protein using the equation 100 − [(( DGCR8 knockdown /β-actin )/( DGCR8 control / β-actin )) × 100]. (C) Changes in endogenous miRNA levels following DGCR8 knockdown were analysed by stemloop RT–PCR analysis. miR-16 is a canonical miRNA control and sno65 is a loading control. Graph shows quantitation of miRNA abundance using the equation: (miRNA experimental condition /sno65)/(miRNA control /sno65). n = 4 for all miRNAs except for miR-16, n = 5; asterisk indicates P ≤ 0.05 (Wilcoxon matched pairs signed-rank test). M indicates a synthetic size marker and filled circle indicates a non-specific primer dimer. ( D ) RT–PCR analysis of Drosha mRNA following expression of TN-Drosha in HEK-293T cells. ( E ) The effect of TN-Drosha expression on endogenous miRNA abundance was analysed by stemloop RT–PCR. Graph shows quantitation of miRNA abundance using the same equation as in C, n = 6; asterick indicates P ≤ 0.05 (Student's t -test). ( F ) Stemloop RT–PCR analysis of minigene-derived miR-877, 1226, 1225, 1228 and endogenous miR-16 isolated from HEK-293T cells transiently transfected with TN-Drosha. sno65 was used as a control. TN-Drosha mRNA expression in HEK-293T cells was analysed by radiolabelled RT–PCR. GAPDH was used as a control. ( G ) Quantitation of miRNA abundance relative to sno65 using the equation: miRNA/sno65. n = 3 for miR-877, 1226 and 1225, n = 5 for miR-1228 and n = 14 for miR-16; * P ≤ 0.05, *** P ≤ 0.0001 (). Data sets were analysed using the Student's t -test with the exception of miR-16, which was analysed using the Wilcoxon matched pairs signed-rank test. In all panels, bars represent the average ± SEM. The horizontal dotted line indicates normalized control levels.

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Control, Quantitation Assay, Marker, Derivative Assay, Isolation, Transfection

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Simtron biogenesis does not require DGCR8, Dicer, Ago2 or XPO5. ( A ) RT–PCR analysis of minigene-derived host gene mRNA and stemloop RT–PCR analysis of minigene-derived miRNA and endogenous miR-16 in Dicer and DGCR8 knockout mouse embryonic stem cells transfected with the wt or splicing-deficient minigene (Δss) or empty vector control (−). sno65 was used as a loading control. Graphs show quantitation of miRNA using the equation: (miRNA experimental condition /sno65)/(miRNA control /sno65). Bars represent the average ± SEM, n = 3. The horizontal dotted lines indicate normalized control levels. ( B ) Stemloop RT–PCR analysis of miR-1225 and miR-1228 immunoprecipitated from HEK-293T cell lysates that were transiently transfected with wt or Δss minigenes, or miR-877 from wt minigene along with pFLAG-Dicer (Dicer) or without (−) and immunoprecipitated with an antibody against the FLAG epitope. Input refers to cell lysates before FLAG immunoprecipitation; Un is the unbound fraction and IP is the immunoprecipitated fraction. Un is 1/20 IP and Input is 1/5 IP. The graph represents the percent of the mature miRNA found in the IP fraction versus the amount that remained in the Un fraction using the equation: (IP/(IP + (Un × 20)) × 100). ( C ) Stemloop RT–PCR analysis of minigene-derived miR-1225, miR-1228 and endogenous miR-16 from Ago2 knockout mouse embryonic fibroblasts. sno65 was used as a loading control. Cells were transiently transfected with wt or Δss minigenes or empty vector control (−). Graph shows quantitation of miRNA abundance using the same equation as in A. Bars represent the average ± SEM, n = 3 and * P ≤ 0.05 or ** P ≤ 0.01, Student's t -test. The horizontal dotted lines indicate normalized control levels. ( D ) Stemloop RT–PCR and RT–PCR analysis of miR-877 (left panel), miR-1225 (middle panel) and miR-1228 (right panel) minigene-expression in HeLa cells following siRNA-directed knockdown of XPO5 . sno65 is a loading control for miRNA using stemloop RT–PCR and GAPDH is a loading control for RT–PCR of XPO5 mRNA.

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Knock-Out, Transfection, Plasmid Preparation, Control, Quantitation Assay, Immunoprecipitation, FLAG-tag, Expressing, Knockdown

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Immunoprecipitation and in vitro processing of simtrons with Drosha. ( A ) Pre-miR-1225 co-immunoprecipitates with Drosha. Pre-miR-1225 derived from wt and Δss minigenes and pre-miR-877 from wt minigene were transiently transfected into HEK-293T cells with pFLAG-Drosha (Drosha) or without (−), and immunoprecipitated with an antibody against the FLAG epitope. Isolated pre-miRNAs were analysed by radiolabelled stemloop RT–PCR and products were separated by 12% native PAGE. Input (In) refers to cell lysates before FLAG immunoprecipitation; Un is the unbound fraction and IP is the immunoprecipitated fraction. Un is 1/20 IP and Input is 1/5 IP. The graph represents the percent of the pre-miRNA found in the IP fraction versus the amount that remained in the Un fraction using the equation: (IP/(IP + (Un × 20)) × 100). ( B ) Drosha-dependent in vitro simtron processing. Radiolabelled RNA transcribed from a PKD1 wt or Δss, ABCF1 wt or pri-miR-16-1 DNA template was incubated with the FLAG-immunoprecipitates from HEK-293T cells, or with HEK-293T WCEs from cells that were not transfected. FLAG-immunoprecipitates were derived from cells transfected with mock transfection (−), pFLAG-GFP (GFP), pFLAG-Drosha (Drosha), pFLAG-Drosha and pFLAG-DGCR8 (Drosha + DGCR8), pFLAG-TN-Drosha (TN Drosha), or FLAG-M2-beads that were incubated with lysis buffer but no cell lysate (−lysate). Template RNA was included as a control (RNA). Reaction products were separated by 8% denaturing PAGE. The sizes of pre-miRNAs are indicated. Asterisk indicates uncharacterized miR-16 cleavage fragments .

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Immunoprecipitation, In Vitro, Derivative Assay, Transfection, FLAG-tag, Isolation, Reverse Transcription Polymerase Chain Reaction, Clear Native PAGE, Incubation, Lysis, Control

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Simtron processing is context independent. ( A ) Diagram comparing intronic and intergenic pre-miRNA expression. ( B ) Control, Dicer (Dicer −/− ) or DGCR8 (DGCR8 −/− ) knockout mouse embryonic stem cells were transiently transfected with the intergenic wt minigene, or intergenic splicing-deficient minigene (Δss). Minigene-derived miRNAs and endogenous miR-16 were analysed by stemloop RT–PCR. Left panel: simtron miR-1225. Right panel: mirtron miR-877. sno65 was analysed as a loading control. ( C ) Graph shows quantitation of miR-1225 abundance using the equation: (miRNA Dicer−/− or DGCR8−/− /sno65)/(miRNA control /sno65). Bars represent the average values ±SEM, n = 4 for Dicer −/− and n = 3 for DGCR8 −/− . The horizontal dotted line indicates normalized control cell levels.

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Expressing, Control, Knock-Out, Transfection, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: Biogenesis of mammalian microRNAs by a non-canonical processing pathway

doi: 10.1093/nar/gks026

Figure Lengend Snippet: Proposed model of simtron biogenesis compared to other miRNA processing pathways. The pathways shown begin with the primary transcript and end with the mature product. Left: simtron pathway, Middle: mirtron pathway, Right: canonical miRNA pathway. Exons are depicted as boxes and introns and miRNAs as lines. Each protein or protein complex is labelled. Proteins labelled with question marks are proposed but not known. Simtrons (such as miR-1225 and miR-1228) processing from the intron involves Drosha and possibly an unknown binding partner. Simtrons are further processed by unknown factors and enter the RISC complex with any of the four human Argonaute proteins. Mirtrons (such as miR-877 and miR-1226) are excised from the host gene by the spliceosome, are debranched, exported from the nucleus by exportin5 (XPO5), cleaved by Dicer and enter the RISC complex. Canonical miRNAs (such as miR-16) are processed by Drosha and DGCR8, exported from the nucleus by XPO5, cleaved by Dicer and enter the RISC complex. All three pathways result in functional miRNAs.

Article Snippet: Hannon) ( ) and DGCR8 knockout (DGCR8 −/− ) cells (Novus Biologicals) were grown on a gelatin layer in Knockout Dulbecco's modified Eagle's medium (Gibco) supplemented with 15% ES cell FBS (Gibco), 1% non-essential amino acids, 1% l -glutamine, 1% penicillin/streptomycin/Amphotericin B, 0.1% ESGRO-LIF and 0.008% beta-mercaptoethanol.

Techniques: Binding Assay, Functional Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification of PSMB5 as a genetic modifier of fragile X-associated tremor/ataxia syndrome.

doi: 10.1073/pnas.2118124119

Figure Lengend Snippet: Fig. 4. Mechanisms of amelioration of CGG-associated toxicity by PSMB5 knockdown. (A and B) PSMB5 knockdown significantly diminishes RAN translation of FMR1 50UTR CGG repeats. Plasmid-transfected NL-based reporters for canonical and RAN translation were expressed in HEK293 cells, following transfec- tion of siRNAs against PSMB5 or nontargeting siRNAs (siNT). Four replicates were performed with consistent results, and the graph represents the pooled data gathered across two replicates (two-way ANOVA with Tukey’s multiple comparisons test; n = 6 per condition; *P ≤0.0332, **P ≤0.0021, ****P ≤ 0.0001). Knockdown of PSMB5 results in significant suppression of RAN translation compared to canonical translation and is not frame-dependent. (C) Sche- matic illustrates a proposed model depicting a potential mechanism of CGG-associated toxicity in FXTAS. DGCR8 normally binds PSMB5 mRNA (Left). In the presence of the expanded premutation FMR1 CGG repeat (Right), DGCR8 is sequestered to the expanded CGG repeat, potentially leading to an increase of PSMB5 mRNA unbound by DGCR8. (D and E) Immunoprecipitation of FLAG-DGCR8 shows less PSMB5 mRNA binds to DGCR8 in the presence of CGG repeats. (D) FLAG-DGCR8 was overexpressed in HEK293T cells for 24 h, then cells were subsequently transfected again with either 50UTR CGG 99x FMR1-EGFP plasmid or pcDNA 3.1. Western blot shows upon immunoprecipitation with anti-FLAG antibody, equal amounts of FLAG-DGCR8 were pulled down and eluted. (E) PSMB5 mRNA bound to FLAG-DGCR8 diminishes significantly in the presence of the expanded CGG repeat. Following immunoprecipitation, the eluent was subject to RNA extraction. Using RT-qPCR, mRNA bound to FLAG-DGCR8 was quantified. Significantly less PSMB5 mRNA was bound to FLAG-DGCR8 in the presence of the CGG repeat. Differences in the levels of U16 snoRNA (also known to be bound by DGCR8) were not statistically significant. Data were pooled across three replicates (two-way ANOVA with Sidak’s multiple comparisons test, n = 3 per condition, **P ≤0.005).

Article Snippet: HEK293T cells transfected with pFLAG/HA DGCR8 (Addgene no. 10921) were grown in 60-mm plates.

Techniques: Knockdown, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, RNA Extraction, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Short intron-derived ncRNAs

doi: 10.1093/nar/gkw1341

Figure Lengend Snippet: Role of Drosha, DGCR8, snRNP70+PRP8, U2AF65+PRP8 and DBR1 in biogenesis of SID. Oligonucleotide arrays developed in , and spotted with the SID that shows a signal above baseline in K562 cells were hybridized with radio-labelled small RNA isolated from K562 cells in which Drosha, DGCR8, snRNP70+PRP8, U2AF65+PRP8, DBR1 or Luc were knocked down ( n = 4). Hybridization signals were plotted as a fold-over baseline signal as described in Material and Methods section. Luc, control luciferase knockdown; Ds, Drosha knockdown; Dg, DGCR8 knockdown; 1+8, snRNP70+PRP8 knockdown; 2+8, U2AF65+PRP8 knockdown; Db, DBR1 knockdown. The hsa-miR-21 (miR21) was used as positive control. Significant differences were assessed using Student's t -test (* P < 0.05). Error bars represent standard error at the mean (SEM).

Article Snippet: pSicoR human Dicer1 (ID 14763), pSicoR human Drosha1 (ID 14766) and pSicoR human DGCR8-1 (ID 14769) were requested from Addgene ( www.addgene.org ).

Techniques: Isolation, Hybridization, Luciferase, Positive Control

Journal: Nucleic Acids Research

Article Title: Short intron-derived ncRNAs

doi: 10.1093/nar/gkw1341

Figure Lengend Snippet: SID interactions with miRNA pathway key factors. ( A ) Native RIP was performed using the indicated antibodies and the effective protein precipitation was confirmed by western blotting. IP, immunoprecipitation; in, 5% input. ( B ) Co-precipitated RNAs were extracted as described in Material and Methods section ( n = 2). Quantitative PCR were performed using specific primer pairs to amplify SID #8, #16, #18, #19, #20, #24, #39, #43, #45 and #46. Amount of immunoprecipitated material is expressed as fold enrichment compared to irrelevant antibody. Ds, Drosha; Dg, DGCR8; Ago, Ago2; Dbr, DBR1; U1, snRNP70; U2, U2AF65. Significant differences with irrelevant antibody were assessed using Student's t -test (* P < 0.05). Error bars represent standard error at the mean (SEM).

Article Snippet: pSicoR human Dicer1 (ID 14763), pSicoR human Drosha1 (ID 14766) and pSicoR human DGCR8-1 (ID 14769) were requested from Addgene ( www.addgene.org ).

Techniques: Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction