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Santa Cruz Biotechnology tbst
Tbst, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/tbst/product/Santa Cruz Biotechnology
Average 92 stars, based on 2 article reviews
Price from $9.99 to $1999.99
tbst - by Bioz Stars, 2020-05
92/100 stars

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Staining:

Article Title: Estrogen induces cell proliferation by promoting ABCG2-mediated efflux in endometrial cancer cells
Article Snippet: .. Blotting membranes were blocked by TBST [150 mM NaCl, 0.1% (v/v) Tween20, and 20 mM Tris] containing 5% (v/v) bovine serum albumin (BSA) and reacted with an anti-ABCG2 antibody (1:250, Santa Cruz Biotechnology, YX, USA) or an anti-β-actin antibody (1:1000, Sigma-Aldrich, Tokyo, Japan) followed by Histofine Simple Stain MAX-PO (Nichirei Bioscience, Tokyo, Japan). ..

Incubation:

Article Title: Licochalcone H induces the apoptosis of human oral squamous cell carcinoma cells via regulation of matrin 3
Article Snippet: .. Following incubation with a horseradish peroxidase (HRP)-labeled secondary antibody against anti-rabbit IgG (LF-SA8002; Ab Frontier, San Diego, CA, USA), anti-goat IgG (cat. no. sc-2020; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-mouse IgG (cat. no. sc-2005; Santa Cruz Biotechnology) was diluted in TBST, the PVDF membrane was reacted with enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and then detected using ImageQuant LAS 4000 Mini system (GE Healthcare Life Sciences, Chalfont, UK) according to the manufacturer's protocol. .. Pull-down assay LCH was coupled to CNBr-activated Sepharose™ 4B matrix beads in 0.1 M NaHCO3 (pH 8.3) containing 0.5 M NaCl overnight at 4°C, according to the manufacturer's protocol.

Article Title: KAT5 Negatively regulates the proliferation of prostate cancer LNCaP cells via the caspase 3-dependent apoptosis pathway
Article Snippet: .. After incubation with 5% skim milk in TBST (20 mM Tris–HCl (pH 7.6), 137 mM NaCl and 0.1% Tween-20) for 1 h, the membrane was washed 3 times with TBST and incubated with antibodies against PARP-1 (1:1000, sc-56197, Santa Cruz), Caspase 3 (1:1000, #9662, Cell Signaling), Bax (1:500, #2772, Cell Signaling), Cytochrome C (1:1000, sc-13156, Santa Cruz), Bcl-w (1:500, sc-11422, Santa Cruz), Bcl-2 (1:500, sc-7382, Santa Cruz), p21 (1:1000, sc-6246, Santa Cruz), β-actin (1:1000, sc-47778, Santa Cruz), KAT5 (1:1000, sc-5725, Santa Cruz) at room temperature. .. Membranes were washed three times for 10 min with TBST and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies for 1 h. Blots were washed with TBST three times and developed with the Western blotting luminol reagent (sc-2048, Santa Cruz) according to the manufacturer’s protocols.

Article Title: Electrical Stimulation with a Conductive Polymer Promotes Neurite Outgrowth and Synaptogenesis in Primary Cortical Neurons in 3D
Article Snippet: .. The membranes were then incubated with synaptophysin, PSD95 (Thermo Fisher Scientific, NSW; dilution factor 1:1000), and PSA-NCAM (Thermo Fisher Scientific, NSW; dilution factor 1:1000) antibodies in TBST containing 1% BSA overnight at 4 o C. Secondary antibodies were anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA; dilution factor 1:5000). .. For visualization, ECL detection reagents were exposed on the GelDoc System (Bio-Rad, Hercules, CA).

Article Title: Lipopolysaccharide from Rhodobacter sphaeroides (TLR4 antagonist) attenuates hypersensitivity and modulates nociceptive factors
Article Snippet: .. Proteins were transferred to Immune-Blot PVDF membranes (Bio-Rad, Hercules, CA) using semi-dry transfer (30 min, 25 V), blocked for 1 h at room temperature using 5% non-fat dry milk (Bio-Rad, Hercules, CA) in Tris-buffered saline with 0.1% Tween 20 (TBST), washed in TBST, and then incubated overnight at 4 °C with primary antibodies targeting the following proteins: TLR4 (Santa Cruz Biotechnology, Santa Cruz, CA #sc-293072, 1:500), IBA-1 (Proteintech #10904-1-AP, 1:1000), GFAP (Santa Cruz Biotechnology, Santa Cruz, CA #sc-6171, 1:1000), IL-1β (Abcam, Cambridge, UK #ab9787, 1:1000), IL-1Ra (Abcam, Cambridge, UK #ab175392, 1:1000), IL-18 (Abcam, Cambridge, UK #ab191860, 1:1000), IL-18BP (Novus Biologicals, Cambridge, UK #NB110-57117, 1:1000), IL-6 (Invitrogen, Carlsbad, CA #ARC0062, 1:500), IL-10 (Invitrogen, Carlsbad, CA #ARC17536, 1:1000), MMP-9 (EMD Millipore, Billerica, MA #AB19016, 1:1000), TIMP-1 (Novus Biologicals, Cambridge, UK #NBP1-96554, 1:1000), and GAPDH (EMD Millipore, Billerica, MA #MAB374, 1:5000; as a loading control). .. Blots were incubated with the corresponding HRP-conjugated secondary polyclonal antibody (Vector Laboratories, Burlingame, CA, peroxidase anti-mouse IgG (H + L) #PI-2000; peroxidase anti-mouse IgG (H + L) #PI-1000, 1:5000) for 1 h at RT.

other:

Article Title: Decreased expression of aquaporin 1 correlates with clinicopathological features of patients with cervical cancer
Article Snippet: The membranes were then washed with 1× TBST, and primary antibodies were detected with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, USA, at a dilution of 1:5,000).

Article Title: Small molecule purine and pseudopurine derivatives: synthesis, cytostatic evaluations and investigation of growth inhibitory effect in non-small cell lung cancer A549
Article Snippet: The next day, membranes were washed in TBST and probed with horseradish peroxidase-conjugated secondary antibodies goat anti-mouse (Santa Cruz Biotechnology) or goat anti-rabbit (Santa Cruz Biotechnology).

Article Title: Role of Hydroxysteroid Dehydrogenase-Like 2 (HSDL2) in Human Ovarian Cancer
Article Snippet: After washing with TBST 3 times, goat anti-mouse IgG coupled to HRP (1: 1000, cat. no. sc-2005, Santa Cruz) as secondary antibody was added and was detected using an EasyBlot ECL kit (cat. no. C506668, Sangon Biotech, Shanghai, China).

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    Santa Cruz Biotechnology tbst mouse monoclonal anti hnrnp a1
    <t>HnRNP</t> A1 binds and remodels the terminal loop of pri-let-7a-1. ( a ) Footprint analysis of the pri-let-7a-1/hnRNP A1 complex. Cleavage patterns were obtained for 5′ 32 P -labeled transcripts (100 × 10 3 /2.5 pmol) incubated in the absence or presence of recombinant hnRNP A1 protein (+, 200 ng) treated with Pb (II)-lead ions (0.5 mM), left panel); or ribonuclease T1 (1.5 units/μl), right panel. F and T identify nucleotide residues subjected to partial digest with formamide (every nucleotide) or ribonuclease T1 (G-specific cleavage), respectively. Electrophoresis was performed in a 10% polyacrylamide gel under denaturing conditions. Positions of selected G residues are indicated. ( b ) Proposed structure of free and hnRNP A1-bound pri-let-7a-1. The sites and intensities of cleavages generated by structure probes (presented below), located at the places of hnRNP A1 binding are shown. Nucleotides are numbered from the 5′ site of Drosha cleavage.
    Tbst Mouse Monoclonal Anti Hnrnp A1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tbst mouse monoclonal anti hnrnp a1/product/Santa Cruz Biotechnology
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    tbst mouse monoclonal anti hnrnp a1 - by Bioz Stars, 2020-05
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    92
    Santa Cruz Biotechnology tbst
    <t>HnRNP</t> A1 binds and remodels the terminal loop of pri-let-7a-1. ( a ) Footprint analysis of the pri-let-7a-1/hnRNP A1 complex. Cleavage patterns were obtained for 5′ 32 P -labeled transcripts (100 × 10 3 /2.5 pmol) incubated in the absence or presence of recombinant hnRNP A1 protein (+, 200 ng) treated with Pb (II)-lead ions (0.5 mM), left panel); or ribonuclease T1 (1.5 units/μl), right panel. F and T identify nucleotide residues subjected to partial digest with formamide (every nucleotide) or ribonuclease T1 (G-specific cleavage), respectively. Electrophoresis was performed in a 10% polyacrylamide gel under denaturing conditions. Positions of selected G residues are indicated. ( b ) Proposed structure of free and hnRNP A1-bound pri-let-7a-1. The sites and intensities of cleavages generated by structure probes (presented below), located at the places of hnRNP A1 binding are shown. Nucleotides are numbered from the 5′ site of Drosha cleavage.
    Tbst, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tbst/product/Santa Cruz Biotechnology
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    tbst - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    93
    Santa Cruz Biotechnology bsa tbst
    <t>HnRNP</t> A1 binds and remodels the terminal loop of pri-let-7a-1. ( a ) Footprint analysis of the pri-let-7a-1/hnRNP A1 complex. Cleavage patterns were obtained for 5′ 32 P -labeled transcripts (100 × 10 3 /2.5 pmol) incubated in the absence or presence of recombinant hnRNP A1 protein (+, 200 ng) treated with Pb (II)-lead ions (0.5 mM), left panel); or ribonuclease T1 (1.5 units/μl), right panel. F and T identify nucleotide residues subjected to partial digest with formamide (every nucleotide) or ribonuclease T1 (G-specific cleavage), respectively. Electrophoresis was performed in a 10% polyacrylamide gel under denaturing conditions. Positions of selected G residues are indicated. ( b ) Proposed structure of free and hnRNP A1-bound pri-let-7a-1. The sites and intensities of cleavages generated by structure probes (presented below), located at the places of hnRNP A1 binding are shown. Nucleotides are numbered from the 5′ site of Drosha cleavage.
    Bsa Tbst, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsa tbst/product/Santa Cruz Biotechnology
    Average 93 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    bsa tbst - by Bioz Stars, 2020-05
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    HnRNP A1 binds and remodels the terminal loop of pri-let-7a-1. ( a ) Footprint analysis of the pri-let-7a-1/hnRNP A1 complex. Cleavage patterns were obtained for 5′ 32 P -labeled transcripts (100 × 10 3 /2.5 pmol) incubated in the absence or presence of recombinant hnRNP A1 protein (+, 200 ng) treated with Pb (II)-lead ions (0.5 mM), left panel); or ribonuclease T1 (1.5 units/μl), right panel. F and T identify nucleotide residues subjected to partial digest with formamide (every nucleotide) or ribonuclease T1 (G-specific cleavage), respectively. Electrophoresis was performed in a 10% polyacrylamide gel under denaturing conditions. Positions of selected G residues are indicated. ( b ) Proposed structure of free and hnRNP A1-bound pri-let-7a-1. The sites and intensities of cleavages generated by structure probes (presented below), located at the places of hnRNP A1 binding are shown. Nucleotides are numbered from the 5′ site of Drosha cleavage.

    Journal: Nature structural & molecular biology

    Article Title: Antagonistic role of hnRNP A1 and KSRP in the regulation of Let-7a biogenesis

    doi: 10.1038/nsmb.1874

    Figure Lengend Snippet: HnRNP A1 binds and remodels the terminal loop of pri-let-7a-1. ( a ) Footprint analysis of the pri-let-7a-1/hnRNP A1 complex. Cleavage patterns were obtained for 5′ 32 P -labeled transcripts (100 × 10 3 /2.5 pmol) incubated in the absence or presence of recombinant hnRNP A1 protein (+, 200 ng) treated with Pb (II)-lead ions (0.5 mM), left panel); or ribonuclease T1 (1.5 units/μl), right panel. F and T identify nucleotide residues subjected to partial digest with formamide (every nucleotide) or ribonuclease T1 (G-specific cleavage), respectively. Electrophoresis was performed in a 10% polyacrylamide gel under denaturing conditions. Positions of selected G residues are indicated. ( b ) Proposed structure of free and hnRNP A1-bound pri-let-7a-1. The sites and intensities of cleavages generated by structure probes (presented below), located at the places of hnRNP A1 binding are shown. Nucleotides are numbered from the 5′ site of Drosha cleavage.

    Article Snippet: Proteins were detected using the following primary antibodies diluted in 1:20 western blocking solution in TBST: mouse monoclonal anti-hnRNP A1 (clone 4B10; 1:1000, Santa Cruz Biotechnology), rabbit polyclonal anti-Lin28a (1:1000, Cell Signalling Technology), mouse monoclonal anti-T7 (1:10,000, Novagen), mouse monoclonal anti-hnRNP L (1:1000, Sigma), mouse-monoclonal anti-KSRP (clone 4C10, 1:500, Sigma), mouse-monoclonal anti-GAPDH (1:10000, Sigma), mouse-monoclonal anti-beta-tubulin (1:10,000, Sigma).

    Techniques: Labeling, Incubation, Recombinant, Electrophoresis, Generated, Binding Assay

    Cartoon displaying the antagonism of KSRP and hnRNP A1 in the post-transcriptional regulation of let-7a processing. ( a ) This model illustrates how the relative levels of KSRP and hnRNP A1 may influence a direct competition between a repressor and an activator binding to the same sequence and determine the processing of let-7a-1 ( b ) Simultaneous occupancy of pri-let-7a-1 terminal loop by both KSRP and hnRNP A1 cannot be ruled out; however, high levels of hnRNP A1 always result in the abrogation of let-7a-1 processing.

    Journal: Nature structural & molecular biology

    Article Title: Antagonistic role of hnRNP A1 and KSRP in the regulation of Let-7a biogenesis

    doi: 10.1038/nsmb.1874

    Figure Lengend Snippet: Cartoon displaying the antagonism of KSRP and hnRNP A1 in the post-transcriptional regulation of let-7a processing. ( a ) This model illustrates how the relative levels of KSRP and hnRNP A1 may influence a direct competition between a repressor and an activator binding to the same sequence and determine the processing of let-7a-1 ( b ) Simultaneous occupancy of pri-let-7a-1 terminal loop by both KSRP and hnRNP A1 cannot be ruled out; however, high levels of hnRNP A1 always result in the abrogation of let-7a-1 processing.

    Article Snippet: Proteins were detected using the following primary antibodies diluted in 1:20 western blocking solution in TBST: mouse monoclonal anti-hnRNP A1 (clone 4B10; 1:1000, Santa Cruz Biotechnology), rabbit polyclonal anti-Lin28a (1:1000, Cell Signalling Technology), mouse monoclonal anti-T7 (1:10,000, Novagen), mouse monoclonal anti-hnRNP L (1:1000, Sigma), mouse-monoclonal anti-KSRP (clone 4C10, 1:500, Sigma), mouse-monoclonal anti-GAPDH (1:10000, Sigma), mouse-monoclonal anti-beta-tubulin (1:10,000, Sigma).

    Techniques: Binding Assay, Sequencing

    KSRP and hnRNP A1 compete for binding to the pri-let-7a-1 terminal loop. ( a ) EMSA analysis of wild-type 5′ 32 P-labeled pri-let-7a-1 transcripts (100 × 10 3 /~2.5 pmol) with GFP-KSRP protein (+, 200 ng) in the absence of presence of various unlabeled RNA competitors (100 pmol). ( b) EMSA analysis of wild-type 5′ 32 P-labeled pri-let-7a-1 transcripts (5 × 10 3 /~0.125 pmol) with GFP-KSRP protein (+, 200 ng, lane 2), UP1 (+, 200 ng, lane 3)), or a constant amount of GFP-KSRP (+, 200 ng) incubated together with increasing amounts of UP1 (+, 200 ng, ++, 400 ng, +++, 800 ng, lanes 4-6). * indicates a slower migrating complex that only appears in the presence of both UP1 and KSRP and could reflect simultaneous occupancy

    Journal: Nature structural & molecular biology

    Article Title: Antagonistic role of hnRNP A1 and KSRP in the regulation of Let-7a biogenesis

    doi: 10.1038/nsmb.1874

    Figure Lengend Snippet: KSRP and hnRNP A1 compete for binding to the pri-let-7a-1 terminal loop. ( a ) EMSA analysis of wild-type 5′ 32 P-labeled pri-let-7a-1 transcripts (100 × 10 3 /~2.5 pmol) with GFP-KSRP protein (+, 200 ng) in the absence of presence of various unlabeled RNA competitors (100 pmol). ( b) EMSA analysis of wild-type 5′ 32 P-labeled pri-let-7a-1 transcripts (5 × 10 3 /~0.125 pmol) with GFP-KSRP protein (+, 200 ng, lane 2), UP1 (+, 200 ng, lane 3)), or a constant amount of GFP-KSRP (+, 200 ng) incubated together with increasing amounts of UP1 (+, 200 ng, ++, 400 ng, +++, 800 ng, lanes 4-6). * indicates a slower migrating complex that only appears in the presence of both UP1 and KSRP and could reflect simultaneous occupancy

    Article Snippet: Proteins were detected using the following primary antibodies diluted in 1:20 western blocking solution in TBST: mouse monoclonal anti-hnRNP A1 (clone 4B10; 1:1000, Santa Cruz Biotechnology), rabbit polyclonal anti-Lin28a (1:1000, Cell Signalling Technology), mouse monoclonal anti-T7 (1:10,000, Novagen), mouse monoclonal anti-hnRNP L (1:1000, Sigma), mouse-monoclonal anti-KSRP (clone 4C10, 1:500, Sigma), mouse-monoclonal anti-GAPDH (1:10000, Sigma), mouse-monoclonal anti-beta-tubulin (1:10,000, Sigma).

    Techniques: Binding Assay, Labeling, Incubation

    Levels of mature let-7a correlate negatively with the levels of hnRNP A1 in human cells. ( a ) Western blot analysis of whole cells extracts from 293T, HeLa and Astrocytoma 1321N1 cells shows different levels of hnRNP A1 expression and similar levels of KSRP, respectively. ( b , c ) Northern blots of total RNA from 293T, HeLa and Astrocytoma 1321N1 cells reveal different levels of mature let-7a. As a loading control, ethidium bromide stain of the RNA is shown on panel b. The black bar on panel c denotes let-7a precursors.

    Journal: Nature structural & molecular biology

    Article Title: Antagonistic role of hnRNP A1 and KSRP in the regulation of Let-7a biogenesis

    doi: 10.1038/nsmb.1874

    Figure Lengend Snippet: Levels of mature let-7a correlate negatively with the levels of hnRNP A1 in human cells. ( a ) Western blot analysis of whole cells extracts from 293T, HeLa and Astrocytoma 1321N1 cells shows different levels of hnRNP A1 expression and similar levels of KSRP, respectively. ( b , c ) Northern blots of total RNA from 293T, HeLa and Astrocytoma 1321N1 cells reveal different levels of mature let-7a. As a loading control, ethidium bromide stain of the RNA is shown on panel b. The black bar on panel c denotes let-7a precursors.

    Article Snippet: Proteins were detected using the following primary antibodies diluted in 1:20 western blocking solution in TBST: mouse monoclonal anti-hnRNP A1 (clone 4B10; 1:1000, Santa Cruz Biotechnology), rabbit polyclonal anti-Lin28a (1:1000, Cell Signalling Technology), mouse monoclonal anti-T7 (1:10,000, Novagen), mouse monoclonal anti-hnRNP L (1:1000, Sigma), mouse-monoclonal anti-KSRP (clone 4C10, 1:500, Sigma), mouse-monoclonal anti-GAPDH (1:10000, Sigma), mouse-monoclonal anti-beta-tubulin (1:10,000, Sigma).

    Techniques: Western Blot, Expressing, Northern Blot, Staining

    Ectopic expression of hnRNP A1 in HeLa and Astrocytoma 1321N1 cells reduces the levels of endogenous mature let-7a. ( b,d ) Northern blots of let-7a using total RNA from HeLa and Astrocytoma cells that have been transiently transfected with a control plasmid (pCG), pCG T7 hnRNP A1, of pCG T7 Lin28a ( a,c ) As loading controls, ethidium bromide stains of total RNA are shown. ( e ) Schematic of the secondary structure of pri-let-7a-1 RNA. Light grey arrows indicate qRT-PCR pair of primers used to detect the levels of pri-let-7a-1 (subjected to Drosha cleavage), whereas black arrows indicate qRT-PCR pair of primers used to detect the levels of upstream region (up.) of pri-let-7a-1 (representing total levels of pri-let-7a-1 transcript) ( f ) Real Time qRT-PCR on RNAs from HeLa cells overexpressing hnRNP A1 reveals a substantial increase in the levels of uncleaved pri-let-7a-1 transcripts (light grey arrows) and at the same time no change in the total levels of pri-let-7a-1 transcript (black arrows). The values were normalized against GAPDH mRNA. The fold change of corresponding RNA fragments abundance mediated by overexpression of either pCG T7 hnRNP A1 of pCG T7 Lin28a was plotted relative to values from a control transfection set to one. Mean values and standard deviations of two independent experiments are shown.

    Journal: Nature structural & molecular biology

    Article Title: Antagonistic role of hnRNP A1 and KSRP in the regulation of Let-7a biogenesis

    doi: 10.1038/nsmb.1874

    Figure Lengend Snippet: Ectopic expression of hnRNP A1 in HeLa and Astrocytoma 1321N1 cells reduces the levels of endogenous mature let-7a. ( b,d ) Northern blots of let-7a using total RNA from HeLa and Astrocytoma cells that have been transiently transfected with a control plasmid (pCG), pCG T7 hnRNP A1, of pCG T7 Lin28a ( a,c ) As loading controls, ethidium bromide stains of total RNA are shown. ( e ) Schematic of the secondary structure of pri-let-7a-1 RNA. Light grey arrows indicate qRT-PCR pair of primers used to detect the levels of pri-let-7a-1 (subjected to Drosha cleavage), whereas black arrows indicate qRT-PCR pair of primers used to detect the levels of upstream region (up.) of pri-let-7a-1 (representing total levels of pri-let-7a-1 transcript) ( f ) Real Time qRT-PCR on RNAs from HeLa cells overexpressing hnRNP A1 reveals a substantial increase in the levels of uncleaved pri-let-7a-1 transcripts (light grey arrows) and at the same time no change in the total levels of pri-let-7a-1 transcript (black arrows). The values were normalized against GAPDH mRNA. The fold change of corresponding RNA fragments abundance mediated by overexpression of either pCG T7 hnRNP A1 of pCG T7 Lin28a was plotted relative to values from a control transfection set to one. Mean values and standard deviations of two independent experiments are shown.

    Article Snippet: Proteins were detected using the following primary antibodies diluted in 1:20 western blocking solution in TBST: mouse monoclonal anti-hnRNP A1 (clone 4B10; 1:1000, Santa Cruz Biotechnology), rabbit polyclonal anti-Lin28a (1:1000, Cell Signalling Technology), mouse monoclonal anti-T7 (1:10,000, Novagen), mouse monoclonal anti-hnRNP L (1:1000, Sigma), mouse-monoclonal anti-KSRP (clone 4C10, 1:500, Sigma), mouse-monoclonal anti-GAPDH (1:10000, Sigma), mouse-monoclonal anti-beta-tubulin (1:10,000, Sigma).

    Techniques: Expressing, Northern Blot, Transfection, Plasmid Preparation, Quantitative RT-PCR, Over Expression

    HnRNP A1 negatively regulates the Drosha-mediated processing of let-7a-1 ( a ) In vitro processing of pri-let-7a-1 is enhanced in the hnRNP A1-depleted extracts. Internally radiolabeled pri-let-7a-1 transcripts (100 × 10 3 c.p.m.) were incubated in the presence of either control HeLa extracts or hnRNP A1-depleted extracts. Lanes (−) shows negative controls with no extract added. Products were analyzed on an 8% polycrylamide gel. Numbers on the left hand side represent RNA size marker. ( b ) In vitro processing of pri-miRNAs pri-let-7a-1 performed in HeLa cell extracts in the presence of increasing amounts of recombinant hnRNP A1 (+, 200 ng, ++, 400ng, +++, 800ng).

    Journal: Nature structural & molecular biology

    Article Title: Antagonistic role of hnRNP A1 and KSRP in the regulation of Let-7a biogenesis

    doi: 10.1038/nsmb.1874

    Figure Lengend Snippet: HnRNP A1 negatively regulates the Drosha-mediated processing of let-7a-1 ( a ) In vitro processing of pri-let-7a-1 is enhanced in the hnRNP A1-depleted extracts. Internally radiolabeled pri-let-7a-1 transcripts (100 × 10 3 c.p.m.) were incubated in the presence of either control HeLa extracts or hnRNP A1-depleted extracts. Lanes (−) shows negative controls with no extract added. Products were analyzed on an 8% polycrylamide gel. Numbers on the left hand side represent RNA size marker. ( b ) In vitro processing of pri-miRNAs pri-let-7a-1 performed in HeLa cell extracts in the presence of increasing amounts of recombinant hnRNP A1 (+, 200 ng, ++, 400ng, +++, 800ng).

    Article Snippet: Proteins were detected using the following primary antibodies diluted in 1:20 western blocking solution in TBST: mouse monoclonal anti-hnRNP A1 (clone 4B10; 1:1000, Santa Cruz Biotechnology), rabbit polyclonal anti-Lin28a (1:1000, Cell Signalling Technology), mouse monoclonal anti-T7 (1:10,000, Novagen), mouse monoclonal anti-hnRNP L (1:1000, Sigma), mouse-monoclonal anti-KSRP (clone 4C10, 1:500, Sigma), mouse-monoclonal anti-GAPDH (1:10000, Sigma), mouse-monoclonal anti-beta-tubulin (1:10,000, Sigma).

    Techniques: In Vitro, Incubation, Marker, Recombinant

    HnRNP A1 specifically binds to the terminal loop of pri-let-7a-1 ( a ) Validated secondary structure of let-7a-1 wild-type and a terminal loop mutant, in which the wild-type terminal loop sequence has been replaced with a GCAA motif (pri-let-7a-1_loop_mt1) ( b ) EMSA analysis of wild-type and a loop mutant sequence (pri-let-7a-1 and pri-let-7a-1_loop_mt1, respectively), with the UP1 protein. Native gel electrophoresis with 5′32 P -labeled transcripts (100 × 10 3 /~2.5 pmol) incubated in the presence of recombinant RRM1, RRM2 or UP1 fragments of hnRNP A1 (200 ng). Let-7a-1 loop indicates unlabeled RNA competitor (100 pmol)

    Journal: Nature structural & molecular biology

    Article Title: Antagonistic role of hnRNP A1 and KSRP in the regulation of Let-7a biogenesis

    doi: 10.1038/nsmb.1874

    Figure Lengend Snippet: HnRNP A1 specifically binds to the terminal loop of pri-let-7a-1 ( a ) Validated secondary structure of let-7a-1 wild-type and a terminal loop mutant, in which the wild-type terminal loop sequence has been replaced with a GCAA motif (pri-let-7a-1_loop_mt1) ( b ) EMSA analysis of wild-type and a loop mutant sequence (pri-let-7a-1 and pri-let-7a-1_loop_mt1, respectively), with the UP1 protein. Native gel electrophoresis with 5′32 P -labeled transcripts (100 × 10 3 /~2.5 pmol) incubated in the presence of recombinant RRM1, RRM2 or UP1 fragments of hnRNP A1 (200 ng). Let-7a-1 loop indicates unlabeled RNA competitor (100 pmol)

    Article Snippet: Proteins were detected using the following primary antibodies diluted in 1:20 western blocking solution in TBST: mouse monoclonal anti-hnRNP A1 (clone 4B10; 1:1000, Santa Cruz Biotechnology), rabbit polyclonal anti-Lin28a (1:1000, Cell Signalling Technology), mouse monoclonal anti-T7 (1:10,000, Novagen), mouse monoclonal anti-hnRNP L (1:1000, Sigma), mouse-monoclonal anti-KSRP (clone 4C10, 1:500, Sigma), mouse-monoclonal anti-GAPDH (1:10000, Sigma), mouse-monoclonal anti-beta-tubulin (1:10,000, Sigma).

    Techniques: Mutagenesis, Sequencing, Nucleic Acid Electrophoresis, Labeling, Incubation, Recombinant