Journal: Plant Physiology

Article Title: Soluble Invertase Expression Is an Early Target of Drought Stress during the Critical, Abortion-Sensitive Phase of Young Ovary Development in Maize 1

doi: 10.1104/pp.005637

Figure Lengend Snippet: In situ localization of Ivr2 soluble invertase mRNAs in tissues of young maize kernels (NK-508) at +6 d post-pollination. Labeled Ivr2 mRNAs are evident in several areas of the maternal tissues that make up the maize kernel at this stage of development, with strongest signal in cells surrounding the tiny, newly fertilized embryo + endosperm (minute and off the field of view; see drawing to lower right) and in cells near vascular bundles of the pedicel. Signal proximal to the embryo + endosperm is localized in basal regions of the nucellus (maternal tissue filling the central kernel during early development), plus cells of the lower pericarp (ovary wall). Individual kernels were sampled as soon as pollinated ovaries could be visually distinguished from non-pollinated neighbors. Fixed and sectioned samples were probed with sense (control, top right) and antisense (in situ localization) RNA probes synthesized with digoxygenin-labeled UTP from a 576-bp Ivr2 fragment as described in the text.

Article Snippet: Ivr1 , Ivr2 , and Incw1 [32 P]ATP probes were produced with a random primed DNA-labeling kit (Roche Diagnostics) whereas for Incw2 , [32 P]UTP RNA probes were generated using a T7 phage polymerase RNA-labeling kit (Ambion Inc., Austin, TX).

Techniques: In Situ, Labeling, Synthesized

Journal: Frontiers in Pharmacology

Article Title: Esomeprazole Increases Airway Surface Liquid pH in Primary Cystic Fibrosis Epithelial Cells

doi: 10.3389/fphar.2018.01462

Figure Lengend Snippet: Chronic esomeprazole does not disrupt epithelial integrity of CF hAECs. (A) Effect of increasing concentrations of apical esomeprazole on TransEpithelial Electrical Resistance (TEER) and Fluorescein Flux measured in Ussing chambers in the presence of a basolateral to apical Cl - gradient ( n = 5, five donors). (B,C) Effect of increasing concentrations of apical eso on resting short-circuit current (Isc, B , n = 5), and (C) amiloride-sensitive Isc (ΔIsc (amil) , black triangle, n = 5), Fsk-induced Isc (ΔIsc (Fsk) , open triangle, n = 5), CFTRinh172-sensitive Isc (ΔIsc (172) , open diamond, n = 5) and UTP-induced Isc (ΔIsc (UTP) , black circles, n = 5).

Article Snippet: Ouabain (O3125), esomeprazole (E7906), N -Acetyl- L -cysteine (A7250), amiloride (A7410), fluorescein (F6377), mitomycin C (M0503), and UTP (U6750) were purchased from Sigma-Aldrich.

Techniques:

Journal: The Journal of Physiology

Article Title: Large‐conductance Ca2+‐activated K+ channel activation by apical P2Y receptor agonists requires hydrocortisone in differentiated airway epithelium

doi: 10.1113/JP274200

Figure Lengend Snippet: Cell model showing the effects of ATP/UTP on specific ion transport processes in control ( A ) and HC0 ( B ) epithelia ATP/UTP stimulate G q ‐coupled purinergic receptors expressed on the apical membrane of surface airway epithelial cells leading to increased [Ca 2+ ] i and activation of PKC. PKC‐dependent phosphorylation of BK permits channel activation after stimulation with ATP/UTP, which also activates Ca 2+ ‐activated chloride channels (ANO1/TMEM16A). In control epithelia, HC suppresses transcription of KCNMA1 that contain the STREX exon. In HC0 epithelia, KCNMA1 STREX mRNAs are the predominant transcript encoding BKα. The STREX exon makes BKα unresponsive to purinergic receptor activation.

Article Snippet: Retinoic acid (RA), benzamil hydrochloride, CFTRinh‐172, phorbol 12‐myristate 13‐acetate (PMA), 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid disodium salt hydrate (DIDS), adenosine 5′‐[γ‐thio]triphosphate tetralithium salt (ATP‐γ‐S), adenosine triphosphate (ATP) and uridine triphosphate (UTP) were purchased from Sigma‐Aldrich Chemical (St Louis, MO, USA).

Techniques: Activation Assay

Journal: Nucleic Acids Research

Article Title: Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy

doi: 10.1093/nar/gkl668

Figure Lengend Snippet: Montage of AFM images of stalled elongation complexes (SEC) and collided complexes. ( A ) OPC formed in the absence of NTPs. ( B ) SEC formed in the presence of ATP, GTP and UTP. ( C ) Collided complexes formed by addition CTP to the reaction mix. Two populations of complexes exist with varying RNAP separations, ones with relatively large, non-contacting separations (top row), and others apparently in contact (bottom row). Image size: 250 × 250 nm.

Article Snippet: In the two-step process, stalled elongation complexes (SECs) were formed by adding ATP, GTP and UTP (Ambion) to a final concentration of 133 μM each.

Techniques: Size-exclusion Chromatography

Journal: Nucleic Acids Research

Article Title: Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy

doi: 10.1093/nar/gkl668

Figure Lengend Snippet: Inter RNAP–RNAP separation distributions along the DNA contour length. RNAP centre separation: ( A ) in the absence of all NTPs (n = 45), i.e. OPCs; ( B ) in the presence of ATP, GTP and UTP only (n = 131), i.e. SECs; ( C ) in the presence of ATP, GTP and CTP, followed by CTP after 20 min ( n = 44); ( D ) formed by addition of all 4 NTPs together ( n = 45) from the OPC state.

Article Snippet: In the two-step process, stalled elongation complexes (SECs) were formed by adding ATP, GTP and UTP (Ambion) to a final concentration of 133 μM each.

Techniques:

Journal: Nucleic Acids Research

Article Title: Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy

doi: 10.1093/nar/gkl668

Figure Lengend Snippet: Tapping mode atomic force microscopy images of convergent transcription complexes. The RNAP are visualised as globular features (blue) on the DNA template (magenta). ( A ) Open promoter complexes (OPCs) formed in the absence of NTPs. DNA molecules are observed with 0, 1 and 2 OPC present. RNAP in OPC are those where the RNAP are roughly one third from either end of the template. ( B ) Stalled elongation complexes (SECs) formed in the presence of ATP, GTP and UTP. RNAP in SEC are recognised as those that are now closer to the centre of the template. ( C ) Elongation complexes formed upon the addition of CTP to the SEC reaction mix. When a DNA molecule contains two SECs, these SECs can collide and sometimes stall against each other. Arrows denote DNA molecules harbouring two RNAP which were used in the statistical analysis of RNAP convergence and collision. Artificial height scale denotes yellow (low) to blue (high): Range = 3 nm.

Article Snippet: In the two-step process, stalled elongation complexes (SECs) were formed by adding ATP, GTP and UTP (Ambion) to a final concentration of 133 μM each.

Techniques: Microscopy, Size-exclusion Chromatography

Journal: Nucleic Acids Research

Article Title: Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy

doi: 10.1093/nar/gkl668

Figure Lengend Snippet: DNA arm contour length distributions: the grey distribution denotes the short arm and black, the long arm. The DNA contour lengths from the apparent centre of the RNAPs to the respective ends of the DNA molecule: ( A ) in the absence of NTPs ( n = 45); ( B ) in the presence of ATP, GTP and UTP only ( n = 45); and incubation with the final NTP, CTP for ( C ) 30 s, ( D ) 10 min and ( E ) 20 min ( n = 25, 30, 45 respectively). The percentages on the right represent the proportion of DNAs within the distribution with 2 RNAP bound, compared to bare DNA and DNA with 1 RNAP bound. The values are normalised to the −NTPs case (panel A).

Article Snippet: In the two-step process, stalled elongation complexes (SECs) were formed by adding ATP, GTP and UTP (Ambion) to a final concentration of 133 μM each.

Techniques: Incubation

Journal: Nucleic Acids Research

Article Title: Phytohormone abscisic acid control RNA-dependent RNA polymerase 6 gene expression and post-transcriptional gene silencing in rice cells

doi: 10.1093/nar/gkm1133

Figure Lengend Snippet: Characterization of RNA polymerization, performed with cell extracts in the presence ( A ) and absence ( B ) of siRNA primers. Sense-stranded RNA corresponding to the internal ICL region (0.5 kb) was used as a template for polymerization in the polymerization mixture containing α- 32 P-UTP. In the presence of ABA (right columns), both primed- and unprimed-polymerization appeared to be induced (see 150 and 500 bp expected polymerization products, respectively), while only unprimed polymerization operated in dsICL-delivered cells in the absence of ABA (B, left).

Article Snippet: For primer-independent polymerization, the reaction was initiated by adding 50 μg of each cell extract to a reaction mixture containing 5 μg of sense-stranded template RNA (ICL), 2 μl polymerization buffer (50 mM HEPES–KOH at pH 7.8, 20 mM NH4 OAc, 5 mM MgCl2 , 0.1 mM EDTA, 0.1% Triton X-100), 0.5 mM ATP, 100 μM CTP, 100 μM GTP, 20 μM UTP, 25 μCi α-32 P-UTP and 0.2 U/μl RNsin (Ambion) in a total volume of 20 μl.

Techniques:

Journal: Purinergic Signalling

Article Title: UTP-induced ATP release is a fine-tuned signalling pathway in osteocytes

doi: 10.1007/s11302-013-9404-1

Figure Lengend Snippet: Analysis of the UTP-induced ATP release pathway. ATP release induced by 10 μM UTP (100 μl/s) was challenged by pharmacological inhibitors. a 10–35 μM CBX had no effect on ATP release, whereas 75 μM

Article Snippet: Cells were exposed to 0.1, 1, 10, 100 or 1,000 μM of the following nucleotide analogues: ADP (Roche, Mannheim, Germany), 2-methyl-thio-ATP (2-meSATP, Sigma, St. Louis, MO), UTP (Roche) and UDP (Sigma).

Techniques:

Journal: Purinergic Signalling

Article Title: UTP-induced ATP release is a fine-tuned signalling pathway in osteocytes

doi: 10.1007/s11302-013-9404-1

Figure Lengend Snippet: UTP induced ATP release. After ten baseline measurements, UTP (0.1, 1 and 10 μM) was injected in the wells leading to slowly increasing and dose-dependent ATP release for the subsequent 7–8 min. Vehicle induced the release

Article Snippet: Cells were exposed to 0.1, 1, 10, 100 or 1,000 μM of the following nucleotide analogues: ADP (Roche, Mannheim, Germany), 2-methyl-thio-ATP (2-meSATP, Sigma, St. Louis, MO), UTP (Roche) and UDP (Sigma).

Techniques: Injection

Journal: Journal of Virology

Article Title: Interaction of Translation Initiation Factor eIF4B with the Poliovirus Internal Ribosome Entry Site

doi:

Figure Lengend Snippet: eIF4B binds to the poliovirus IRES RNA. (A) Detection of eIF4B in RRL. UV cross-linking assays were performed with poliovirus IRES RNA labeled with either [α- 32 P]ATP, -CTP, -GTP or -UTP as indicated. Proteins were resolved on SDS-10% polyacrylamide gels and visualized by autoradiography. Molecular masses of marker proteins are indicated. (B) Immunoprecipitation of eIF4B. The binding reaction was performed with RRL and [α- 32 P]CTP-labeled IRES RNA. After the UV cross-linking reaction, eIF4B was immunoprecipitated (IP) with anti-eIF4B antiserum (α-4B) (lane 1). Preimmune serum was used as negative control (pre.) (lane 2). Ten percent of the proteins from the supernatants (sup.) (right panel) was concentrated by TCA-precipitation (lanes 3 and 4), resolved on SDS-10% polyacrylamide gels, and visualized by autoradiography. 4B, eIF4B. (C) No effect of La protein on eIF4B binding. Poliovirus IRES RNA was used in the UV cross-linking assay by using RRL either without (lane 1) or after adding increasing amounts of recombinant La protein (rec. La) as indicated (lanes 2 to 7). (D and E) Immunoblots with anti-La antibodies 4B6 (D) and 3B9 (E). The bands representing endogenous La (end. La) and added recombinant La (rec. La) are indicated.

Article Snippet: Labeled RNAs were synthesized as described previously ( ) by using SP6 RNA polymerase in the presence of 2.5 μM [α-32 P]CTP, -ATP, -GTP, or -UTP (400 Ci/mmol; Amersham) as indicated with 15 μM nonradioactive labeling nucleotide added.

Techniques: Labeling, Autoradiography, Marker, Immunoprecipitation, Binding Assay, Negative Control, TCA Precipitation, Recombinant, Western Blot

Journal: Journal of Virology

Article Title: Splicing Regulatory Elements within tat Exon 2 of Human Immunodeficiency Virus Type 1 (HIV-1) Are Characteristic of Group M but Not Group O HIV-1 Strains

doi:

Figure Lengend Snippet: Mutagenesis of rev 3′ splice site A4e of HS1-A70 does not increase splicing at the env/nef 3′ splice site A5. (A) Denaturing PAGE (6% gel) of [ 32 P]UTP-labeled HS1-A70 and rev A4e splice site mutant (HS1-A4e) substrates spliced in vitro. Also shown is in vitro splicing of NL4-3 substrate HS1-X. HS1-A4e is an AG-to-AC mutant at the A4e 3′ splice site. The positions of precursors and products spliced at the indicated splice sites are shown. (B) Quantitation of spliced tat , rev , and env/nef spliced products from the in vitro-spliced substrates shown in panel A. Multiple gels were quantitated, and the amounts of products spliced at the tat , rev , and env/nef 3′ splice sites were calculated based on of uridine content of the different RNA species.

Article Snippet: In vitro runoff RNA transcripts labeled with [32 P]UTP (Amersham Pharmacia Biotech) were carried out as previously described ( ).

Techniques: Mutagenesis, Polyacrylamide Gel Electrophoresis, Labeling, In Vitro, Quantitation Assay

Journal: Journal of Virology

Article Title: Splicing Regulatory Elements within tat Exon 2 of Human Immunodeficiency Virus Type 1 (HIV-1) Are Characteristic of Group M but Not Group O HIV-1 Strains

doi:

Figure Lengend Snippet: Comparison of tat 3′ splice site usage in different HIV-1 strains by in vitro splicing assays. (A) Denaturing PAGE of [ 32 P]UTP-labeled HIV-1 substrates spliced in vitro. The positions of precursors, spliced products, and tat lariat products are indicated. On the left is a 6% gel comparing HS1-X, HS1-SF2, and HS1-A70; on the right is a 4% gel comparing HS1-ESS4 and HS1-A70. HS1-A70 spliced product migrates more slowly because the restriction site used to produce the linearized DNA template for transcription of RNA substrates is 7 nt further downstream than for HS1-X and HS1-SF2, resulting in longer RNA products. tat lariats migrate more slowly than the linear RNA species on 6% compared to 4% gels. (B) Quantitation of spliced tat RNA from the in vitro-spliced substrates shown in panel A. Multiple gels were quantitated, and the amounts of product spliced at the tat 3′ splice site (A3) were calculated based on uridine content of the RNA species.

Article Snippet: In vitro runoff RNA transcripts labeled with [32 P]UTP (Amersham Pharmacia Biotech) were carried out as previously described ( ).

Techniques: In Vitro, Polyacrylamide Gel Electrophoresis, Labeling, Quantitation Assay

Journal: Journal of Virology

Article Title: Splicing Regulatory Elements within tat Exon 2 of Human Immunodeficiency Virus Type 1 (HIV-1) Are Characteristic of Group M but Not Group O HIV-1 Strains

doi:

Figure Lengend Snippet: Mutagenesis of the rev A4b 3′ AG dinucleotide enhances splicing of the env/nef 3′ splice site in HS1-SF2. (A) Denaturing PAGE (6% gel) of [ 32 P]UTP-labeled HS1-SF2 and rev A4b splice site mutant (HS1-SF4b) substrates spliced in vitro. Also shown is in vitro splicing of NL4-3 substrate HS1-X. HS1-SF4b is an AG-to-AC mutant at the A4b 3′ splice site. The positions of precursors and products spliced at the indicated splice sites are shown. (B) Quantitation of spliced tat , rev , and env/nef spliced products from the in vitro-spliced substrates shown in panel A. Multiple gels were quantitated, and the amounts of products spliced at the tat , rev , and env/nef 3′ splice sites were calculated based on of uridine content of the different RNA species.

Article Snippet: In vitro runoff RNA transcripts labeled with [32 P]UTP (Amersham Pharmacia Biotech) were carried out as previously described ( ).

Techniques: Mutagenesis, Polyacrylamide Gel Electrophoresis, Labeling, In Vitro, Quantitation Assay

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: In Vitro System for Coupling RNAP II Transcription to Primary microRNA Processing and a Three-way System for RNAP II Transcription/Splicing/microRNA Processing

doi: 10.1007/978-1-4939-8624-8_4

Figure Lengend Snippet: Coupled RNAP II transcription/splicing/pri-miRNA processing in vitro. (A) Structure of the CMV-FTZ-let DNA template. The sizes of FTZ pre-mRNA exons, intron, 5’ flanking region, 3’ flanking region, and pre-let-7a are indicated. The thick line indicates pri-miRNA sequences and the thin lines indicate intron sequences. (B) CMV-FTZ-let DNA template was incubated in nuclear extract for 20 min to assemble a pre-initiation complex followed by addition of 32 P-UTP, ATP, and Creatine Phosphate and continued incubation for 10 min. α-Amanitin was added to stop transcription and incubation was continued for the indicated times. Full length transcript, spliced mRNA, exon1, pre-miRNAs and 5’/3’ flanks are indicated. Markers (in base pairs) are indicated and Ori indicates the gel origin. Samples were run for 1 hour. (C) Same as (B), except that samples were run for 2 hours to obtain a better separation of pre-mRNA and 5’/3’ flanks.

Article Snippet: For assembly of pre-initiation complexes (PICs), incubate the CMV-let-7a DNA template (4 μls containing 800 ng DNA) with 1 μl MgCl2 (80 mM), 3 μl 15% polyvinyl alcohol (PVA) and 15 μl HeLa nuclear extract ( see ) at 30°C for 20 min. To initiate RNAP II transcription, add 1 μl 32 P-UTP (800 Ci/mmol; Perkin Elmer Life Sciences), 1 μl ATP (12.5 mM) and 1 μl Creatine Phosphate di-Tris salt (500 mM) to the reaction mixture.

Techniques: In Vitro, Incubation

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: In Vitro System for Coupling RNAP II Transcription to Primary microRNA Processing and a Three-way System for RNAP II Transcription/Splicing/microRNA Processing

doi: 10.1007/978-1-4939-8624-8_4

Figure Lengend Snippet: Coupled RNAP II transcription/pri-miRNA processing in vitro. (A) Structure of the CMV-let-7a DNA template. The sizes of 5’ flanking region, 3’ flanking region, and pre-let-7a are indicated. The thick line indicates the natural pri-miRNA sequences and the thin lines indicate the vector sequences. (B) CMV-let-7a DNA template was incubated in nuclear extract for 20 min to assemble a pre-initiation complex followed by addition of 32 P-UTP, ATP, and Creatine Phosphate and continued incubation for 5 min. α-Amanitin was added to stop transcription and incubation was continued for the indicated times. Pri-miRNAs, pre-miRNAs and 5’/3’ flanks are indicated. The endogenous U6 snRNA and tRNA in the extract are labeled by 32 Markers (in base pairs) are indicated, and Ori indicates the gel origin. (C) A dark exposure of (B).

Article Snippet: For assembly of pre-initiation complexes (PICs), incubate the CMV-let-7a DNA template (4 μls containing 800 ng DNA) with 1 μl MgCl2 (80 mM), 3 μl 15% polyvinyl alcohol (PVA) and 15 μl HeLa nuclear extract ( see ) at 30°C for 20 min. To initiate RNAP II transcription, add 1 μl 32 P-UTP (800 Ci/mmol; Perkin Elmer Life Sciences), 1 μl ATP (12.5 mM) and 1 μl Creatine Phosphate di-Tris salt (500 mM) to the reaction mixture.

Techniques: In Vitro, Plasmid Preparation, Incubation, Labeling

Journal: PLoS Genetics

Article Title: Ectodysplasin/NF-κB Promotes Mammary Cell Fate via Wnt/β-catenin Pathway

doi: 10.1371/journal.pgen.1005676

Figure Lengend Snippet: Edar expression and NF-κB reporter expression co-localize in the mammary epithelium. (A) Edar transcripts were detected by in situ hybridization with a 35 S-UTP-labeled probe in mammary buds at E12.5 and E13.5. (B) NF-κB reporter was initially expressed throughout the mammary bud in control embryos, but became localized to the basal layer of the epithelium around E13.5. (C) In K14-Eda embryos, reporter activity stayed high throughout the mammary bud at E12.5 and E13.5. mb4 = mammary bud number 4. (D) The supernumerary buds exhibited mosaic expression of the reporter which was less pronounced in the in the neck (left) than in mammary primordia forming between buds 3 and 4 (right). (Scale bar: 100 μm.)

Article Snippet: Radioactive in situ hybridization on paraffin sections was carried out according to previously described protocols using 35 S-UTP labelled (Amersham) probe specific to Edar [ ].

Techniques: Expressing, In Situ Hybridization, Labeling, Activity Assay

Journal: The American Journal of Pathology

Article Title: Distinct Role of Fibroblast Growth Factor-2 and Vascular Endothelial Growth Factor on Tumor Growth and Angiogenesis

doi:

Figure Lengend Snippet: VEGF and FGF-2 expression by Tet-FGF-2 and AS-VEGF/Tet-FGF-2 transfectants. Tet-FGF-2 15H cells were transfected with the pcDNA-3 expression vector harboring the VEGF 121 anti-sense cDNA. A: Stable transfectants (AS-VEGF/Tet-FGF-2 A5 and C1 clones) and mock-transfected cells (Tet-FGF-2 A4 and C7 clones) were analyzed for AS-VEGF mRNA steady-state levels by Northern blotting of total RNA (20 μg/lane) using a digoxigenin-UTP-labeled AS-VEGF riboprobe. B: Uniform loading of the gel was assessed by methylene blue staining of the filter. C: Serum-free conditioned media from the different clones (60 μg of protein) were probed with anti-VEGF antibodies by Western blotting showing decreased levels of the secreted forms of VEGF in AS-VEGF/Tet-FGF-2 cells. D: Lysates (500 μg) from cells grown in the absence (−) or in the presence (+) of tetracycline were loaded onto 0.1-ml heparin-Sepharose columns and bound material was probed with anti-FGF-2 antibodies in a Western blot. All of the clones express the 24-, 22-, and 18-kd FGF-2 isoforms whose expression is hampered by tetracycline treatment. E: Endothelial GM 7373 cells were incubated for 24 hours with increasing concentrations of the conditioned media from untreated and tetracycline-treated (+tet) Tet-FGF-2 A4 and AS-VEGF/Tet-FGF-2 C1 clones. At the end of incubation cells were trypsinized and counted. Data are the mean ± SE of four determinations.

Article Snippet: Blots were hybridized with riboprobes to AS-VEGF mRNA labeled with digoxigenin-UTP by in vitro transcription with T7 RNA polymerase DIG RNA labeling kit (Roche Applied Science, Basel, Switzerland).

Techniques: Expressing, Transfection, Plasmid Preparation, Northern Blot, Labeling, Staining, Clone Assay, Western Blot, Incubation

Journal: Arthritis Research

Article Title: Kinesin-like protein CENP-E is upregulated in rheumatoid synovial fibroblasts

doi:

Figure Lengend Snippet: Demonstration of CENP-E messenger RNA and protein in rheumatoid synovium by in situ hybridization and immunohistochemistry. (a, b) A digoxigenin-labelled RNA probe transcribed from the amplified RNA arbitrarily primed-PCR gene product is used on rheumatoid arthritis snap-frozen sections. Double-labelling for CENP-E messenger RNA (black staining), and alkaline phosphatase antialkaline phosphatase counterstaining using anti-fibroblast antibodies (red staining) shows CENP-E expression in numerous fibroblasts througout the synovium (a, arrows). (b) The intensive CENP-E messenger RNA expression in two synovial fibroblasts. (c) Shows numerous fibroblasts expressing CENP-E protein [black staining (arrows), counterstaining with fast red]. Original magnifications × 300 (a, c) and × 600 (b).

Article Snippet: Probes were labelled with digoxigenin-uridine triphosphate (Boehringer Mannheim).

Techniques: In Situ Hybridization, Immunohistochemistry, Amplification, Polymerase Chain Reaction, Staining, Expressing, RNA Expression

Journal: Nucleic Acids Research

Article Title: Far upstream element binding protein 1 binds the internal ribosomal entry site of enterovirus 71 and enhances viral translation and viral growth

doi: 10.1093/nar/gkr682

Figure Lengend Snippet: FBP1 associates with EV71 5′-UTR. ( A ) FBP1 association with EV71 5′-UTR was confirmed by competition assay and western blot. The biotinylated RNA association proteins were loaded to SDS–PAGE (12%). FBP1 antibody was utilized in this western blot. Various amounts of unlabeled EV71 5′-UTR and yeast tRNA RNA probe were added to compete with the biotinylated EV71 5′-UTR probe interacting with FBP1 in RD cell lysate. Lanes 1 and 6 contained cell lysate (200 µg) only. An unlabeled EV71 5′-UTR RNA probe was used in the competition assay (lanes 3–5), and an unlableled yeast tRNA probe was utilized (lanes 8–10). ( B ) EV71 5′-UTR associates with cellular protein FBP1 in the various cell lines, SK-N-MC, SF268, RD and Vero cell. Cell lysates are shown in lanes 1, 6, 11 and 16. Various cell extracts were incubated in the absence of RNA (lanes 2, 7, 12 and 17) or in the presence of biotin-16-UTP only (lanes 3, 8, 13 and 18), non-biotinylated EV71 5′-UTR (lanes 4, 9, 14 and 19) or biotinylated EV71 5′-UTR (lanes 5, 10, 15 and 20). After the streptavidin beads were washed, the EV71 5′-UTR associated proteins were detected using SDS–PAGE (12%). FBP1 protein was analyzed by western blot with anti-FBP1 cellular protein antibody.

Article Snippet: A biotinylated RNA probe synthesized in a 20 µl MEGAscript transcription reaction by adding 1.25 µl 20 mM biotinylated UTP, Biotin-16-UTP (Roche).

Techniques: Competitive Binding Assay, Western Blot, SDS Page, Incubation

Journal: Frontiers in Plant Science

Article Title: The Digestive System of the Two-Spotted Spider Mite, Tetranychus urticae Koch, in the Context of the Mite-Plant Interaction

doi: 10.3389/fpls.2018.01206

Figure Lengend Snippet: Permeability of ventriculus-posterior midgut barrier in spider mites. (A–D) Serial transverse sections of the dorsal region of spider mite body in the area of connection between ventriculus (v) and posterior midgut (pm). (A) Typical arrangement of V-shaped posterior midgut positioned between two caudal caeca. LC, large cells of caudal caeca epithelium adjacent to posterior midgut (pm); MC, microvilli cells of posterior midgut (yellow arrowhead). (B,C) Anteriorward sections through the posterior midgut that narrows, while the posterior-dorsal portion of the ventriculus appears (red arrowhead). (D) Section through the point of ventriculus-posterior midgut connection. (E) Distribution of the 50 nm fluorescent microspheres and fluorescent dye Alexa Fluor 555 (1250 Da) in mite midgut upon the ingestion of their mixture. Alexa Fluor 555 readily passes through midgut and accumulates within posterior midgut, while 50 nm microspheres are retained in the caeca lumen. (F) Distribution of the FITC-labeled dextrans of different sizes and erioglaucine dye in mite midgut upon the ingestion of their mixture. Erioglaucine (793 Da) and fluorescein-12-UTP (1034 Da) accumulate in posterior midgut while FITC-labeled dextrans (4, 40, and 500 kDa) are retained in the caeca lumen. Guanine autofluorescence can be seen within digestive cells in midgut and in pellets in the posterior midgut. Scale bars: (A–D) : 25 μm; (E,F) : 100 μm.

Article Snippet: To ensure the intactness of fluorescein-labeled dextran, the fluorescein-12-UTP (Roche) was delivered as a control.

Techniques: Permeability, Labeling