Structured Review

TaKaRa human circakt3 cdna
<t>circAKT3</t> expression is increased in CDDP-resistant GC cells and tissues. a Validated expression of 10 circRNAs in the tissues from 44 GC patients using RT-qPCR. b Expression levels of circAKT3 in CDDP-resistant and their matched sensitive parental cell lines (SGC7901CDDP, BGC823CDDP, SGC7901 and BGC823) normalized to GAPDH expression. c The existence of circAKT3 was validated by Sanger sequencing. The red arrow shows the “head-to-tail” splicing sites of circAKT3. d The existence of circAKT3 was validated in SGC7901CDDP and BGC823CDDP cell lines by RT-PCR. Divergent primers amplified circAKT3 in <t>cDNA</t> but not in genomic DNA (gDNA). GAPDH served as a negative control. e RNA from SGC7901CDDP and BGC823CDDP cells was treated with or without RNase R for RT-qPCR. The relative levels of circAKT3 and AKT3 mRNA were normalized to the values measured in the mock-treated cells. f Levels of small nucleolar RNA (U6, as a positive control for the nuclear fraction), GAPDH (positive control for cytoplasmic fraction), AKT3 mRNA and circRNAs from the nuclear and cytoplasmic fractions of SGC7901CDDP cells. g RNA stability of circular and linear transcripts of AKT3 and of 18S rRNA in SGC7901CDDP cells. h Representative images of RNA FISH of circAKT3 expression in SGC7901CDDP cells, which show that circAKT3 is predominantly localized to the cytoplasm. Nuclei were stained with DAPI. Scale bar, 10 μm. The results are presented as the mean ± SEM. * P
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Images

1) Product Images from "Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression"

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression

Journal: Molecular Cancer

doi: 10.1186/s12943-019-0969-3

circAKT3 expression is increased in CDDP-resistant GC cells and tissues. a Validated expression of 10 circRNAs in the tissues from 44 GC patients using RT-qPCR. b Expression levels of circAKT3 in CDDP-resistant and their matched sensitive parental cell lines (SGC7901CDDP, BGC823CDDP, SGC7901 and BGC823) normalized to GAPDH expression. c The existence of circAKT3 was validated by Sanger sequencing. The red arrow shows the “head-to-tail” splicing sites of circAKT3. d The existence of circAKT3 was validated in SGC7901CDDP and BGC823CDDP cell lines by RT-PCR. Divergent primers amplified circAKT3 in cDNA but not in genomic DNA (gDNA). GAPDH served as a negative control. e RNA from SGC7901CDDP and BGC823CDDP cells was treated with or without RNase R for RT-qPCR. The relative levels of circAKT3 and AKT3 mRNA were normalized to the values measured in the mock-treated cells. f Levels of small nucleolar RNA (U6, as a positive control for the nuclear fraction), GAPDH (positive control for cytoplasmic fraction), AKT3 mRNA and circRNAs from the nuclear and cytoplasmic fractions of SGC7901CDDP cells. g RNA stability of circular and linear transcripts of AKT3 and of 18S rRNA in SGC7901CDDP cells. h Representative images of RNA FISH of circAKT3 expression in SGC7901CDDP cells, which show that circAKT3 is predominantly localized to the cytoplasm. Nuclei were stained with DAPI. Scale bar, 10 μm. The results are presented as the mean ± SEM. * P
Figure Legend Snippet: circAKT3 expression is increased in CDDP-resistant GC cells and tissues. a Validated expression of 10 circRNAs in the tissues from 44 GC patients using RT-qPCR. b Expression levels of circAKT3 in CDDP-resistant and their matched sensitive parental cell lines (SGC7901CDDP, BGC823CDDP, SGC7901 and BGC823) normalized to GAPDH expression. c The existence of circAKT3 was validated by Sanger sequencing. The red arrow shows the “head-to-tail” splicing sites of circAKT3. d The existence of circAKT3 was validated in SGC7901CDDP and BGC823CDDP cell lines by RT-PCR. Divergent primers amplified circAKT3 in cDNA but not in genomic DNA (gDNA). GAPDH served as a negative control. e RNA from SGC7901CDDP and BGC823CDDP cells was treated with or without RNase R for RT-qPCR. The relative levels of circAKT3 and AKT3 mRNA were normalized to the values measured in the mock-treated cells. f Levels of small nucleolar RNA (U6, as a positive control for the nuclear fraction), GAPDH (positive control for cytoplasmic fraction), AKT3 mRNA and circRNAs from the nuclear and cytoplasmic fractions of SGC7901CDDP cells. g RNA stability of circular and linear transcripts of AKT3 and of 18S rRNA in SGC7901CDDP cells. h Representative images of RNA FISH of circAKT3 expression in SGC7901CDDP cells, which show that circAKT3 is predominantly localized to the cytoplasm. Nuclei were stained with DAPI. Scale bar, 10 μm. The results are presented as the mean ± SEM. * P

Techniques Used: Expressing, Quantitative RT-PCR, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Positive Control, Fluorescence In Situ Hybridization, Staining

2) Product Images from "Puerarin protects brain tissue against cerebral ischemia/reperfusion injury by inhibiting the inflammatory response"

Article Title: Puerarin protects brain tissue against cerebral ischemia/reperfusion injury by inhibiting the inflammatory response

Journal: Neural Regeneration Research

doi: 10.4103/1673-5374.147934

Detection of mRNA expression of Toll-like receptor 4, myeloid differentiation factor 88, nuclear factor kappa B and tumor necrosis factor-α in ischemic brain tissue by real-time reverse transcription-PCR (RT-PCR)
Figure Legend Snippet: Detection of mRNA expression of Toll-like receptor 4, myeloid differentiation factor 88, nuclear factor kappa B and tumor necrosis factor-α in ischemic brain tissue by real-time reverse transcription-PCR (RT-PCR)

Techniques Used: Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

3) Product Images from "Sur8 mediates tumorigenesis and metastasis in colorectal cancer"

Article Title: Sur8 mediates tumorigenesis and metastasis in colorectal cancer

Journal: Experimental & Molecular Medicine

doi: 10.1038/emm.2016.58

Effect of Sur8 modulation on ERK and Akt activities, and proliferation of CRC cells. ( a ) Different stable CRC cells infected with either InshCon or InshSur8 lentivirus were treated with Dox for 72 h. WCLs were immunoblotted with the indicated antibodies. ( b , c ) The indicated stable CRC cells infected with either InshCon or InshSur8 ( b ), and with InConOE or InSur8OE lentivirus ( c ), were seeded in 12-well plates. Cells were treated with fresh Dox every 24 h to induce Sur8 knockdown ( b ) or overexpression ( c ) and cell growth was measured at different time points as described in the Materials and Methods section. The data shown are the mean±s.d. of three independent experiments. CRC, colorectal cancer; Dox, doxycycline; ERK, extracellular signal-regulated kinase; IHC, immunohistochemistry; WCLs, whole-cell lysates.
Figure Legend Snippet: Effect of Sur8 modulation on ERK and Akt activities, and proliferation of CRC cells. ( a ) Different stable CRC cells infected with either InshCon or InshSur8 lentivirus were treated with Dox for 72 h. WCLs were immunoblotted with the indicated antibodies. ( b , c ) The indicated stable CRC cells infected with either InshCon or InshSur8 ( b ), and with InConOE or InSur8OE lentivirus ( c ), were seeded in 12-well plates. Cells were treated with fresh Dox every 24 h to induce Sur8 knockdown ( b ) or overexpression ( c ) and cell growth was measured at different time points as described in the Materials and Methods section. The data shown are the mean±s.d. of three independent experiments. CRC, colorectal cancer; Dox, doxycycline; ERK, extracellular signal-regulated kinase; IHC, immunohistochemistry; WCLs, whole-cell lysates.

Techniques Used: Infection, Over Expression, Immunohistochemistry

Effect of Sur8 knockdown or overexpression on the transforming potential of CRC cells. ( a , b ) Stable HCT116 or LoVo cells infected with either InshCon or InshSur8 lentivirus were seeded in six-well plates and treated with fresh Dox every 24 h for 14 days to induce Sur8 knockdown. Foci were stained with crystal violet, and images were captured. ( c , d ) Stable CCD18Co and SW480 cells infected with either InConOE or InSur8OE lentivirus were seeded in 24-well plates for performing the anchorage-independent soft agar assay. Cells were treated with fresh Dox every 24 h to induce Sur8 overexpression, and images of the colonies were captured after 3 weeks. The data shown are the mean±s.d. of three independent experiments. *** P
Figure Legend Snippet: Effect of Sur8 knockdown or overexpression on the transforming potential of CRC cells. ( a , b ) Stable HCT116 or LoVo cells infected with either InshCon or InshSur8 lentivirus were seeded in six-well plates and treated with fresh Dox every 24 h for 14 days to induce Sur8 knockdown. Foci were stained with crystal violet, and images were captured. ( c , d ) Stable CCD18Co and SW480 cells infected with either InConOE or InSur8OE lentivirus were seeded in 24-well plates for performing the anchorage-independent soft agar assay. Cells were treated with fresh Dox every 24 h to induce Sur8 overexpression, and images of the colonies were captured after 3 weeks. The data shown are the mean±s.d. of three independent experiments. *** P

Techniques Used: Over Expression, Infection, Staining, Soft Agar Assay

4) Product Images from "HnRNP C, YB-1 and hnRNP L coordinately enhance skipping of human MUSK exon 10 to generate a Wnt-insensitive MuSK isoform"

Article Title: HnRNP C, YB-1 and hnRNP L coordinately enhance skipping of human MUSK exon 10 to generate a Wnt-insensitive MuSK isoform

Journal: Scientific Reports

doi: 10.1038/srep06841

HnRNP C, YB-1 and hnRNP L bind to exon 10 of human MUSK . (a, b) Mouse nucleotides are introduced into ESS5 (a) and ESS12 (b) of pH-wt to generate pH-mB5 and pH-mB12, respectively. RT-PCR of each mutated minigene in HeLa cells is compared with that of pH-wt. Primer positions are shown by arrows. (c) Sequences of ESS5 RNA probes carrying human (H-B5), mouse (m-B5), and partially deleted (H-B5Δ5) sequences. (d) 32 P-labeled H-B5 or m-B5 RNA probe was incubated in the presence or absence of HeLa nuclear extract (NuEX) and resolved on a native polyacrylamide gel to observe free and protein-bound RNA species (arrows). (e) Coomassie blue staining of RNA affinity-purified products from HeLa nuclear extract using the indicated biotinylated RNA probes. Three proteins of ~70, ~50, and ~40 kDa (arrows) are differentially associated with H-B5 compare to m-B5 and H-B5Δ5. Mass spectrometry analysis revealed that the three proteins are HnRNP L (L), YB-1, and hnRNP C (C). (f) Immunoblotting (IB) of RNA affinity purified proteins in panel (e) with the indicated antibodies.
Figure Legend Snippet: HnRNP C, YB-1 and hnRNP L bind to exon 10 of human MUSK . (a, b) Mouse nucleotides are introduced into ESS5 (a) and ESS12 (b) of pH-wt to generate pH-mB5 and pH-mB12, respectively. RT-PCR of each mutated minigene in HeLa cells is compared with that of pH-wt. Primer positions are shown by arrows. (c) Sequences of ESS5 RNA probes carrying human (H-B5), mouse (m-B5), and partially deleted (H-B5Δ5) sequences. (d) 32 P-labeled H-B5 or m-B5 RNA probe was incubated in the presence or absence of HeLa nuclear extract (NuEX) and resolved on a native polyacrylamide gel to observe free and protein-bound RNA species (arrows). (e) Coomassie blue staining of RNA affinity-purified products from HeLa nuclear extract using the indicated biotinylated RNA probes. Three proteins of ~70, ~50, and ~40 kDa (arrows) are differentially associated with H-B5 compare to m-B5 and H-B5Δ5. Mass spectrometry analysis revealed that the three proteins are HnRNP L (L), YB-1, and hnRNP C (C). (f) Immunoblotting (IB) of RNA affinity purified proteins in panel (e) with the indicated antibodies.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Labeling, Incubation, Staining, Affinity Purification, Mass Spectrometry

Binding of hnRNP C, YB-1, and hnRNP L to specific motifs enhances coordinated skipping of MUSK exon 10. (a, b) Scanning mutagenesis to map the binding motifs of hnRNP C, YB-1, and hnRNP L in ESS5 (H-B5). RNA probe sequences are shown in the upper panels, where discordant nucleotides between human and mouse are underlined in H-B5 (ESS5). Artificial mutations are shown in red. RNA affinity-purified products are detected by immunoblotting in the lower panels. The results are indicated on the right side by “+” and “−” for positive and negative binding to each probe, respectively. (c) The resulting binding site of each factor from panels (a) and (b) is schematically shown. Essential binding nucleotides are indicated by large green letters and are underlined. (d) Schematic of a reporter minigene (pSPL3-human- MUSK -MS2-PP7). MS2 coat protein-binding hairpin RNA (blue) is substituted for the first half of ESS5 (binding site of hnRNP C). Similarly, PP7 coat protein-binding hairpin RNA (orange) is substituted for the second half of ESS5 (binding sites of hnRNP L and YB-1). (e) RT-PCR of pSPL3-human- MUSK -MS2-PP7 minigene in HeLa cells that are co-transfected with the indicated effectors. Blue and orange letters match to those in (d). The mean and SD ( n = 3) of the ratio of exon skipping in each treatment is shown below the gel image.
Figure Legend Snippet: Binding of hnRNP C, YB-1, and hnRNP L to specific motifs enhances coordinated skipping of MUSK exon 10. (a, b) Scanning mutagenesis to map the binding motifs of hnRNP C, YB-1, and hnRNP L in ESS5 (H-B5). RNA probe sequences are shown in the upper panels, where discordant nucleotides between human and mouse are underlined in H-B5 (ESS5). Artificial mutations are shown in red. RNA affinity-purified products are detected by immunoblotting in the lower panels. The results are indicated on the right side by “+” and “−” for positive and negative binding to each probe, respectively. (c) The resulting binding site of each factor from panels (a) and (b) is schematically shown. Essential binding nucleotides are indicated by large green letters and are underlined. (d) Schematic of a reporter minigene (pSPL3-human- MUSK -MS2-PP7). MS2 coat protein-binding hairpin RNA (blue) is substituted for the first half of ESS5 (binding site of hnRNP C). Similarly, PP7 coat protein-binding hairpin RNA (orange) is substituted for the second half of ESS5 (binding sites of hnRNP L and YB-1). (e) RT-PCR of pSPL3-human- MUSK -MS2-PP7 minigene in HeLa cells that are co-transfected with the indicated effectors. Blue and orange letters match to those in (d). The mean and SD ( n = 3) of the ratio of exon skipping in each treatment is shown below the gel image.

Techniques Used: Binding Assay, Mutagenesis, Affinity Purification, Protein Binding, Reverse Transcription Polymerase Chain Reaction, Transfection

HnRNP C, YB-1, and hnRNP L coordinately promote skipping of MUSK exon 10. (a) Immunoblotting (IB) using the indicated antibodies after gene knockdown with siRNA against control (siCont), hnRNP C (siC), YB-1 (siYB-1), and hnRNP L (siL) in HeLa cells. (b) RT-PCR of pSPL3-human- MUSK (pH-wt) minigene in HeLa cells treated with the indicated siRNAs. The mean and SD ( n = 3) of the ratio of exon skipping in each treatment is shown below the gel image. (c) Immunoblotting (IB) with the indicated antibodies after cDNA overexpression of hnRNP C, YB-1, and hnRNP L in HeLa cells. Endogenous (endo) and exogenous (exon) YB-1 proteins are pointed by arrows. (d) RT-PCR of pH-wt minigene in HeLa cells co-transfected with the indicated cDNAs. The mean and SD ( n = 3) of the ratio of exon skipping in each treatment is shown below the gel image.
Figure Legend Snippet: HnRNP C, YB-1, and hnRNP L coordinately promote skipping of MUSK exon 10. (a) Immunoblotting (IB) using the indicated antibodies after gene knockdown with siRNA against control (siCont), hnRNP C (siC), YB-1 (siYB-1), and hnRNP L (siL) in HeLa cells. (b) RT-PCR of pSPL3-human- MUSK (pH-wt) minigene in HeLa cells treated with the indicated siRNAs. The mean and SD ( n = 3) of the ratio of exon skipping in each treatment is shown below the gel image. (c) Immunoblotting (IB) with the indicated antibodies after cDNA overexpression of hnRNP C, YB-1, and hnRNP L in HeLa cells. Endogenous (endo) and exogenous (exon) YB-1 proteins are pointed by arrows. (d) RT-PCR of pH-wt minigene in HeLa cells co-transfected with the indicated cDNAs. The mean and SD ( n = 3) of the ratio of exon skipping in each treatment is shown below the gel image.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Over Expression, Transfection

5) Product Images from "PTTG1 regulated by miR-146a-3p promotes bladder cancer migration, invasion, metastasis and growth"

Article Title: PTTG1 regulated by miR-146a-3p promotes bladder cancer migration, invasion, metastasis and growth

Journal: Oncotarget

doi: 10.18632/oncotarget.13507

miR-146a-3p was downregulated in BC cells and tissues ( A ) Real-time RT-PCR analysis suggested miR-146a-3p was downregulated in BC tissues compared to the adjacent normal bladder tissues. ( B ) miR-146a-3p levels were negatively correlated with PTTG1 levels in BC tissues. ( C ) Real-time RT-PCR analysis suggested miR-146a-3p was downregulated in another eight BC tissues compared to the adjacent normal bladder tissues. ( D ) Real-time RT-PCR analysis suggested miR-146a-3p was downregulated in BC cells. ( E ) Real-time RT-PCR analysis suggested miR-146a-3p overexpression inhibited PTTG1 mRNA levels. ( F ) Western blot assay suggested miR-146a-3p overexpression decreased PTTG1 protein levels. GAPDH was used as the loading control. * P
Figure Legend Snippet: miR-146a-3p was downregulated in BC cells and tissues ( A ) Real-time RT-PCR analysis suggested miR-146a-3p was downregulated in BC tissues compared to the adjacent normal bladder tissues. ( B ) miR-146a-3p levels were negatively correlated with PTTG1 levels in BC tissues. ( C ) Real-time RT-PCR analysis suggested miR-146a-3p was downregulated in another eight BC tissues compared to the adjacent normal bladder tissues. ( D ) Real-time RT-PCR analysis suggested miR-146a-3p was downregulated in BC cells. ( E ) Real-time RT-PCR analysis suggested miR-146a-3p overexpression inhibited PTTG1 mRNA levels. ( F ) Western blot assay suggested miR-146a-3p overexpression decreased PTTG1 protein levels. GAPDH was used as the loading control. * P

Techniques Used: Quantitative RT-PCR, Over Expression, Western Blot

6) Product Images from "A lateral flow dipstick combined with reverse transcription recombinase polymerase amplification for rapid and visual detection of the bovine respirovirus 3"

Article Title: A lateral flow dipstick combined with reverse transcription recombinase polymerase amplification for rapid and visual detection of the bovine respirovirus 3

Journal: Molecular and Cellular Probes

doi: 10.1016/j.mcp.2018.08.004

Testing the BPIV3 LFD RT-RPA assay for analytical specificity. (A) Specificity of the LFD RT-RPA assay. RPA products detected using LFD assay yield visually positive results only when tested using cDNA synthesis from BPIV3; results are visually negative for all other bovine viral pathogens with similar clinical symptoms. Samples were tested in triplicate with one reaction displayed in figure for each triplicate. (B) The results of amplification products of the LFD RT-RPA on 2% agarose gel. Lanes 1 to 6: BEFV, VSV, BcoV, BRSV, BVDV, and IBRV, respectively; Lane 7: positive control of BPIV3. (C) Quality detection of RNA/DNA of BEFV, VSV, BcoV, BRSV, BVDV, IBRV and BPIV3. The RNA/DNA of different virus was prepared for specificity detection by PCR reaction with viral specific primers. The positive amplification results were shown in Lane 2, Lane 4, Lane 6, Lane 8, Lane 10, Lane 12, Lane 14, respectively. Lane 1, Lane 3, Lane 5, Lane 7, Lane 9, Lane 11, Lane 13 were negative controls with DNase-free water as template.
Figure Legend Snippet: Testing the BPIV3 LFD RT-RPA assay for analytical specificity. (A) Specificity of the LFD RT-RPA assay. RPA products detected using LFD assay yield visually positive results only when tested using cDNA synthesis from BPIV3; results are visually negative for all other bovine viral pathogens with similar clinical symptoms. Samples were tested in triplicate with one reaction displayed in figure for each triplicate. (B) The results of amplification products of the LFD RT-RPA on 2% agarose gel. Lanes 1 to 6: BEFV, VSV, BcoV, BRSV, BVDV, and IBRV, respectively; Lane 7: positive control of BPIV3. (C) Quality detection of RNA/DNA of BEFV, VSV, BcoV, BRSV, BVDV, IBRV and BPIV3. The RNA/DNA of different virus was prepared for specificity detection by PCR reaction with viral specific primers. The positive amplification results were shown in Lane 2, Lane 4, Lane 6, Lane 8, Lane 10, Lane 12, Lane 14, respectively. Lane 1, Lane 3, Lane 5, Lane 7, Lane 9, Lane 11, Lane 13 were negative controls with DNase-free water as template.

Techniques Used: RT RPA Assay, Recombinase Polymerase Amplification, Amplification, Agarose Gel Electrophoresis, Positive Control, Polymerase Chain Reaction

7) Product Images from "De novo Synthesis of SAA1 in the Placenta Participates in Parturition"

Article Title: De novo Synthesis of SAA1 in the Placenta Participates in Parturition

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.01038

Induction of proinflammatory factors in placenta and fetal membranes by intraperitoneal administration of LPS or SAA1 in the mouse. (A,B) Induction of SAA1/2 expression in mouse placenta (A) and fetal membranes (B) by intraperitoneal administration of LPS (5 mg/kg BW, 6 h) at gestational day 16.5 ( n = 3). (C–E) Induction of IL-1β (C) , TNF-α (D) and COX-2 (E) in the mouse placenta by intraperitoneal administration of LPS (5 mg/kg BW, 6 h) and SAA1 (8 μg/kg BW, 6 h) at gestational day 16.5 ( n = 4). Data are mean ± SEM. * P
Figure Legend Snippet: Induction of proinflammatory factors in placenta and fetal membranes by intraperitoneal administration of LPS or SAA1 in the mouse. (A,B) Induction of SAA1/2 expression in mouse placenta (A) and fetal membranes (B) by intraperitoneal administration of LPS (5 mg/kg BW, 6 h) at gestational day 16.5 ( n = 3). (C–E) Induction of IL-1β (C) , TNF-α (D) and COX-2 (E) in the mouse placenta by intraperitoneal administration of LPS (5 mg/kg BW, 6 h) and SAA1 (8 μg/kg BW, 6 h) at gestational day 16.5 ( n = 4). Data are mean ± SEM. * P

Techniques Used: Expressing

8) Product Images from "Survey of single-nucleotide polymorphisms in the gene encoding human deoxyribonuclease I-like 2 producing loss of function potentially implicated in the pathogenesis of parakeratosis"

Article Title: Survey of single-nucleotide polymorphisms in the gene encoding human deoxyribonuclease I-like 2 producing loss of function potentially implicated in the pathogenesis of parakeratosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0175083

All of the 38 non-synonymous SNPs predicted to be “probably damaging (score = 1.000) in the human DNase 1L2 gene.
Figure Legend Snippet: All of the 38 non-synonymous SNPs predicted to be “probably damaging (score = 1.000) in the human DNase 1L2 gene.

Techniques Used:

All the SNPs originated from frameshift/nonsense mutations in the DNase 1L2 gene. The position of the amino acid residue, in the codon of which mutations occur, are shown on the precursor of the DNase 1L2 protein presented as a solid bar. When the DNase 1L2 activities of the conditioned medium from the cells transfected with the constructs corresponding to each mutation marked with solid arrow were determined using the SRED method [ 16 ], no activity from all the construct examined could be detected under our assay conditions. SNPs marked with asterisk are generated by nonsense mutation.
Figure Legend Snippet: All the SNPs originated from frameshift/nonsense mutations in the DNase 1L2 gene. The position of the amino acid residue, in the codon of which mutations occur, are shown on the precursor of the DNase 1L2 protein presented as a solid bar. When the DNase 1L2 activities of the conditioned medium from the cells transfected with the constructs corresponding to each mutation marked with solid arrow were determined using the SRED method [ 16 ], no activity from all the construct examined could be detected under our assay conditions. SNPs marked with asterisk are generated by nonsense mutation.

Techniques Used: Transfection, Construct, Mutagenesis, Activity Assay, Generated

Effect of the amino acid substitution derived from each non-synonymous SNPs examined on the DNase 1L2 activity.
Figure Legend Snippet: Effect of the amino acid substitution derived from each non-synonymous SNPs examined on the DNase 1L2 activity.

Techniques Used: Derivative Assay, Activity Assay

Effect of deletion of the proline-rich domain from the DNase 1L2 protein on the activity.
Figure Legend Snippet: Effect of deletion of the proline-rich domain from the DNase 1L2 protein on the activity.

Techniques Used: Activity Assay

9) Product Images from "Long non-coding RNA X-inactive specific transcript silencing ameliorates primary graft dysfunction following lung transplantation through microRNA-21-dependent mechanism"

Article Title: Long non-coding RNA X-inactive specific transcript silencing ameliorates primary graft dysfunction following lung transplantation through microRNA-21-dependent mechanism

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2019.102600

Schematic representation of the regulatory network of XIST/miR-21/IL-12A during the NET formation in PGD following lung transplantation. XIST, as a ceRNA of miR-21, upregulates the expression of IL-12A, which induces NET formation and ultimately accelerates PGD after lung transplantation.
Figure Legend Snippet: Schematic representation of the regulatory network of XIST/miR-21/IL-12A during the NET formation in PGD following lung transplantation. XIST, as a ceRNA of miR-21, upregulates the expression of IL-12A, which induces NET formation and ultimately accelerates PGD after lung transplantation.

Techniques Used: Transplantation Assay, Expressing

LncRNA XIST silencing decreases IL-12A expression to suppress NET formation in PMNs in vitro by elevating miR-21. a, the content of NET-DNA was elevated in PMA-treated PMNs after transfection with sh-XIST and IL-12A overexpression vector, as determined by ELISA; b, the content of free DNA in PMNs was increased after transfection with sh-XIST and IL-12A overexpression vector, as detected by immunofluorescence staining; c, the apoptosis rate of PMA-treated PMNs was promoted after transfection with sh-XIST and IL-12A overexpression vector, as determined by flow cytometry. All data were presented as mean ± standard deviation. Comparison of data among multiple groups was conducted using one-way ANOVA; the experiment was repeated three times; * p
Figure Legend Snippet: LncRNA XIST silencing decreases IL-12A expression to suppress NET formation in PMNs in vitro by elevating miR-21. a, the content of NET-DNA was elevated in PMA-treated PMNs after transfection with sh-XIST and IL-12A overexpression vector, as determined by ELISA; b, the content of free DNA in PMNs was increased after transfection with sh-XIST and IL-12A overexpression vector, as detected by immunofluorescence staining; c, the apoptosis rate of PMA-treated PMNs was promoted after transfection with sh-XIST and IL-12A overexpression vector, as determined by flow cytometry. All data were presented as mean ± standard deviation. Comparison of data among multiple groups was conducted using one-way ANOVA; the experiment was repeated three times; * p

Techniques Used: Expressing, In Vitro, Transfection, Over Expression, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Flow Cytometry, Standard Deviation

LncRNA XIST upregulates IL-12A by binding to miR-21. a, there were binding sites between miR-21 and XIST, according to online prediction; b, the luciferase activity of XIST-wt was inhibited by miR-21 mimic, as confirmed by the dual luciferase reporter assay; c, XIST was pulled-down by miR-21-Mut in RNA pull-down assay; d, the expression of miR-21 and XIST coprecipitated with Ago2 magnetic beads was enhanced in the RIP assay; e, XIST was upregulated in BALF of PGD patients, as measured by RT-qPCR; f, the expression of miR-21 was promoted and IL-12A expression was suppressed in PMNs after the suppression of XIST, as measured by RT-qPCR; g, there was a negative correlation between miR-21 and XIST, as determined by the correlation analysis; h, IL-12A and XIST were positively correlated, as determined by the correlation analysis. All data were presented as mean ± standard deviation. The data between PGD patients ( n = 24) and non-PGD patients ( n = 18) in panel e were compared and analysed using unpaired t -test; one-way analysis of variance followed by Tukey's post hoc test was used to analyze the variance of multiple groups with normal distribution. Brown-Forsythe and Welch analysis of variance, followed by Tamhane's post hoc test, was used to analyze the variance of multiple groups with unequal variances; the experiment was repeated three times; * p
Figure Legend Snippet: LncRNA XIST upregulates IL-12A by binding to miR-21. a, there were binding sites between miR-21 and XIST, according to online prediction; b, the luciferase activity of XIST-wt was inhibited by miR-21 mimic, as confirmed by the dual luciferase reporter assay; c, XIST was pulled-down by miR-21-Mut in RNA pull-down assay; d, the expression of miR-21 and XIST coprecipitated with Ago2 magnetic beads was enhanced in the RIP assay; e, XIST was upregulated in BALF of PGD patients, as measured by RT-qPCR; f, the expression of miR-21 was promoted and IL-12A expression was suppressed in PMNs after the suppression of XIST, as measured by RT-qPCR; g, there was a negative correlation between miR-21 and XIST, as determined by the correlation analysis; h, IL-12A and XIST were positively correlated, as determined by the correlation analysis. All data were presented as mean ± standard deviation. The data between PGD patients ( n = 24) and non-PGD patients ( n = 18) in panel e were compared and analysed using unpaired t -test; one-way analysis of variance followed by Tukey's post hoc test was used to analyze the variance of multiple groups with normal distribution. Brown-Forsythe and Welch analysis of variance, followed by Tamhane's post hoc test, was used to analyze the variance of multiple groups with unequal variances; the experiment was repeated three times; * p

Techniques Used: Binding Assay, Luciferase, Activity Assay, Reporter Assay, Pull Down Assay, Expressing, Magnetic Beads, Quantitative RT-PCR, Standard Deviation

Silencing of XIST inhibits the expression of IL-12A by upregulating miR-21 to suppress NET formation and ultimately to ameliorate PGD after lung transplantation. a, XIST was poorly expressed before and during the single lung transplantation, whilst it was overexpressed after single lung transplantation where * p
Figure Legend Snippet: Silencing of XIST inhibits the expression of IL-12A by upregulating miR-21 to suppress NET formation and ultimately to ameliorate PGD after lung transplantation. a, XIST was poorly expressed before and during the single lung transplantation, whilst it was overexpressed after single lung transplantation where * p

Techniques Used: Expressing, Transplantation Assay

IL-12A is a target gene of miR-21, and can reverse the inhibitory effect of miR-21 on NET formation. a, there were binding sites between miR-21 and IL-12A, as predicted on the TargetScan software ( http://www.targetscan.org/ ); b, IL-12A-wt expression was repressed by miR-21 mimic verified by the dual luciferase reporter assay; c, the mRNA expression of IL-12A in PMNs following transfection with miR-21 mimic was inhibited, as detected by RT-qPCR; d, the protein expression of IL-12A was suppressed in response to the treatment with miR-21 mimic, as determined by western blot analysis; e, the protein expression of IL-12A in BALF of PGD patients was enhanced, as determined by western blot analysis; f, the mRNA expression of IL-12A in BALF of PGD patients was facilitated, as determined by RT-qPCR; g, the content of NET-DNA in PMA-treated PMNs transfected with sh-IL-12A or miR-21 mimic was reduced, as assessed by ELISA; h, the apoptosis rate was promoted in PMA-treated PMNs transfected with sh-IL-12A or miR-21 mimic, as detected by flow cytometry; i, the mRNA expression of IL-12A in BALF of rats was diminished after miR-21 mimic treatment, as determined by RT-qPCR; j, the content of free DNA was decreased in PMNs transfected with sh-IL-12A or miR-21 mimic, as detected by immunofluorescence staining (400 ×). All data were presented as mean ± standard deviation. The data between two groups were compared and analysed using unpaired t -test; one-way analysis of variance followed by Tukey's post hoc test was used to analyze the variance of multiple groups with normal distribution. Brown-Forsythe and Welch analysis of variance, followed by Tamhane's post hoc test, was used to analyze the variance of multiple groups with unequal variances; the experiment was repeated three times; in panel f, n = 18 (non-PGD patients); n = 24 (PGD patients); * p
Figure Legend Snippet: IL-12A is a target gene of miR-21, and can reverse the inhibitory effect of miR-21 on NET formation. a, there were binding sites between miR-21 and IL-12A, as predicted on the TargetScan software ( http://www.targetscan.org/ ); b, IL-12A-wt expression was repressed by miR-21 mimic verified by the dual luciferase reporter assay; c, the mRNA expression of IL-12A in PMNs following transfection with miR-21 mimic was inhibited, as detected by RT-qPCR; d, the protein expression of IL-12A was suppressed in response to the treatment with miR-21 mimic, as determined by western blot analysis; e, the protein expression of IL-12A in BALF of PGD patients was enhanced, as determined by western blot analysis; f, the mRNA expression of IL-12A in BALF of PGD patients was facilitated, as determined by RT-qPCR; g, the content of NET-DNA in PMA-treated PMNs transfected with sh-IL-12A or miR-21 mimic was reduced, as assessed by ELISA; h, the apoptosis rate was promoted in PMA-treated PMNs transfected with sh-IL-12A or miR-21 mimic, as detected by flow cytometry; i, the mRNA expression of IL-12A in BALF of rats was diminished after miR-21 mimic treatment, as determined by RT-qPCR; j, the content of free DNA was decreased in PMNs transfected with sh-IL-12A or miR-21 mimic, as detected by immunofluorescence staining (400 ×). All data were presented as mean ± standard deviation. The data between two groups were compared and analysed using unpaired t -test; one-way analysis of variance followed by Tukey's post hoc test was used to analyze the variance of multiple groups with normal distribution. Brown-Forsythe and Welch analysis of variance, followed by Tamhane's post hoc test, was used to analyze the variance of multiple groups with unequal variances; the experiment was repeated three times; in panel f, n = 18 (non-PGD patients); n = 24 (PGD patients); * p

Techniques Used: Binding Assay, Software, Expressing, Luciferase, Reporter Assay, Transfection, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunofluorescence, Staining, Standard Deviation

10) Product Images from "Sperm flagella protein components: Human meichroacidin constructed by the membrane occupation and recognition nexus motif"

Article Title: Sperm flagella protein components: Human meichroacidin constructed by the membrane occupation and recognition nexus motif

Journal: Reproductive Medicine and Biology

doi: 10.1111/j.1447-0578.2005.00108.x

Analysis of mRNA in various tissues. (a) The human multiple tissue Northern blot was hybridized with the full‐length h‐MCA cDNA. (b) Reverse transcription polymerase chain reaction analysis using a Rapid‐Scan gene expression panel containing cDNA from 24 different human tissues. Arrow indicates the single signal included three kinds of h‐MCA cDNA splicing variants. The numbers in the right margin indicate the lengths of size marker fragments (bp). The expression of actin mRNA was also examined as a control.
Figure Legend Snippet: Analysis of mRNA in various tissues. (a) The human multiple tissue Northern blot was hybridized with the full‐length h‐MCA cDNA. (b) Reverse transcription polymerase chain reaction analysis using a Rapid‐Scan gene expression panel containing cDNA from 24 different human tissues. Arrow indicates the single signal included three kinds of h‐MCA cDNA splicing variants. The numbers in the right margin indicate the lengths of size marker fragments (bp). The expression of actin mRNA was also examined as a control.

Techniques Used: Northern Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Marker

11) Product Images from "AtREM1, a Member of a New Family of B3 Domain-Containing Genes, Is Preferentially Expressed in Reproductive Meristems 1"

Article Title: AtREM1, a Member of a New Family of B3 Domain-Containing Genes, Is Preferentially Expressed in Reproductive Meristems 1

Journal: Plant Physiology

doi: 10.1104/pp.010323

Expression analysis of REM1 . A, An RNA blot containing 1.5 μg of poly(A) + RNA from different organs of Arabidopsis was hybridized with the cDNA REM1 and a probe corresponding to ribosomal protein L3 as a control for gel loading ( RBP4 ). B, Expression analysis of AtREM1 and AP2 by reverse transcriptase (RT)-PCR in different organs from adult plants. RT-PCR products were blotted onto membranes and hybridized with the corresponding probes. C, RT-PCR analysis of expression of REM1 in apices from young seedlings at different stages of development. C, Cotyledon and hypocotyl from young seedlings; IA, inflorescence apex; IF, immature flower, before anthesis; L, leaf; MF, flower at anthesis; R, root; S, stem; Sq, silique.
Figure Legend Snippet: Expression analysis of REM1 . A, An RNA blot containing 1.5 μg of poly(A) + RNA from different organs of Arabidopsis was hybridized with the cDNA REM1 and a probe corresponding to ribosomal protein L3 as a control for gel loading ( RBP4 ). B, Expression analysis of AtREM1 and AP2 by reverse transcriptase (RT)-PCR in different organs from adult plants. RT-PCR products were blotted onto membranes and hybridized with the corresponding probes. C, RT-PCR analysis of expression of REM1 in apices from young seedlings at different stages of development. C, Cotyledon and hypocotyl from young seedlings; IA, inflorescence apex; IF, immature flower, before anthesis; L, leaf; MF, flower at anthesis; R, root; S, stem; Sq, silique.

Techniques Used: Expressing, Northern blot, Reverse Transcription Polymerase Chain Reaction, IA

12) Product Images from "Hypoxia stimulates insulin-like growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: A possible model for IGFBP-1 expression in fetal hypoxia"

Article Title: Hypoxia stimulates insulin-like growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: A possible model for IGFBP-1 expression in fetal hypoxia

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

doi:

Hypoxia induction of IGFBP-1 in HepG2 cells. ( A ) Cells were grown in serum-containing medium in the presence of 20% and 2% O 2 , respectively. After 24 hr, the conditioned medium was collected and assayed in triplicate for IGFBP-1 using an immunoradiometric assay. Values shown are the mean ± SEM of six different experiments. ( B ) Time course of hypoxia experiments, with media being harvested after 2, 6, 12, and 24 hr of exposure of HepG2 cells at 2% and 20% pO 2 . Data represent the mean of four experiments run in triplicate and are reported as “fold induction.” ( C ) At the end of each time period, total RNA was collected from the cells and a 1.5-kb mRNA transcript was detected by Northern blot analysis using an Eco RI fragment (938 bp) of the IGFBP-1 cDNA. 18S rRNA shown at the bottom. ( D ) Densitometry of Northern blot shown in C , with fold increase normalized to 18S rRNA.
Figure Legend Snippet: Hypoxia induction of IGFBP-1 in HepG2 cells. ( A ) Cells were grown in serum-containing medium in the presence of 20% and 2% O 2 , respectively. After 24 hr, the conditioned medium was collected and assayed in triplicate for IGFBP-1 using an immunoradiometric assay. Values shown are the mean ± SEM of six different experiments. ( B ) Time course of hypoxia experiments, with media being harvested after 2, 6, 12, and 24 hr of exposure of HepG2 cells at 2% and 20% pO 2 . Data represent the mean of four experiments run in triplicate and are reported as “fold induction.” ( C ) At the end of each time period, total RNA was collected from the cells and a 1.5-kb mRNA transcript was detected by Northern blot analysis using an Eco RI fragment (938 bp) of the IGFBP-1 cDNA. 18S rRNA shown at the bottom. ( D ) Densitometry of Northern blot shown in C , with fold increase normalized to 18S rRNA.

Techniques Used: Immunoradiometric Assay, Northern Blot

Specificity of IGF-binding protein response to hypoxia. ( A ) Autoradiogram of representative Western ligand blot of media conditioned by HepG2 cells cultured under normoxia (20% O 2 , lanes d–h) and hypoxia (2% O 2 , lanes i–m) for time (in hr) shown. Controls are in lanes a–c. Nonpregnant human serum (NPS, lane a) demonstrates IGFBP-3, a prominent doublet with relative molecular mass between 38 and 43 kDa. Lane b shows IGFBP-2 (34 kDa) and IGFBP-4 (24 kDa) in human seminal plasma (SP). Human midgestation amniotic fluid (AF, lane c) shows IGFBP-1 (28 kDa). Molecular mass markers are shown on the right in kDa and migration of IGFBPs is shown on the left. ( B ) Autoradiogram of representative Northern blot of total RNA isolated from HepG2 cells cultured 24 hr in normoxia (20%) or hypoxia at % O 2 shown. Membrane was probed sequentially with 32 P-labeled cDNA probes (see text and Materials and Methods ). Transcript sizes are shown on the right in kb. Exposure times were: for IGFBP-1, 2 hr; IGFBP-2, 3 days; IGFBP-3, 30 days; IGFBP-4, 3 days; IGFBP-5 and IGFBP-6, 7 days; 18S, 16 hr.
Figure Legend Snippet: Specificity of IGF-binding protein response to hypoxia. ( A ) Autoradiogram of representative Western ligand blot of media conditioned by HepG2 cells cultured under normoxia (20% O 2 , lanes d–h) and hypoxia (2% O 2 , lanes i–m) for time (in hr) shown. Controls are in lanes a–c. Nonpregnant human serum (NPS, lane a) demonstrates IGFBP-3, a prominent doublet with relative molecular mass between 38 and 43 kDa. Lane b shows IGFBP-2 (34 kDa) and IGFBP-4 (24 kDa) in human seminal plasma (SP). Human midgestation amniotic fluid (AF, lane c) shows IGFBP-1 (28 kDa). Molecular mass markers are shown on the right in kDa and migration of IGFBPs is shown on the left. ( B ) Autoradiogram of representative Northern blot of total RNA isolated from HepG2 cells cultured 24 hr in normoxia (20%) or hypoxia at % O 2 shown. Membrane was probed sequentially with 32 P-labeled cDNA probes (see text and Materials and Methods ). Transcript sizes are shown on the right in kb. Exposure times were: for IGFBP-1, 2 hr; IGFBP-2, 3 days; IGFBP-3, 30 days; IGFBP-4, 3 days; IGFBP-5 and IGFBP-6, 7 days; 18S, 16 hr.

Techniques Used: Binding Assay, Western Blot, Cell Culture, Migration, Northern Blot, Isolation, Labeling

13) Product Images from "Expression and Function of Sex Pheromones and Receptors in the Homothallic Ascomycete Gibberella zeae "

Article Title: Expression and Function of Sex Pheromones and Receptors in the Homothallic Ascomycete Gibberella zeae

Journal: Eukaryotic Cell

doi: 10.1128/EC.00272-07

Analysis of transcript levels of pheromone precursor genes ppg1 and ppg2 and pheromone receptor genes pre1 and pre2 in G. zeae . Total RNA was extracted from 3-day-old fresh mycelia without induction (NI), 3-day-old mycelia after induction (IM), or ascospores (AS). Twenty micrograms was subjected to Northern analysis with the PCR product of the target gene serving as a probe (left panel), and 1 μg was used to synthesize first-strand RNA cDNA for RT-PCR (right panel).
Figure Legend Snippet: Analysis of transcript levels of pheromone precursor genes ppg1 and ppg2 and pheromone receptor genes pre1 and pre2 in G. zeae . Total RNA was extracted from 3-day-old fresh mycelia without induction (NI), 3-day-old mycelia after induction (IM), or ascospores (AS). Twenty micrograms was subjected to Northern analysis with the PCR product of the target gene serving as a probe (left panel), and 1 μg was used to synthesize first-strand RNA cDNA for RT-PCR (right panel).

Techniques Used: Northern Blot, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

14) Product Images from "Influence of simulated microgravity on the activation of the small GTPase Rho involved in cytoskeletal formation - molecular cloning and sequencing of bovine leukemia-associated guanine nucleotide exchange factor"

Article Title: Influence of simulated microgravity on the activation of the small GTPase Rho involved in cytoskeletal formation - molecular cloning and sequencing of bovine leukemia-associated guanine nucleotide exchange factor

Journal: BMC Biochemistry

doi: 10.1186/1471-2091-7-19

Real-time quantitative PCR . The expression of LARG mRNA was measured by real-time quantitative PCR as described in the Methods section. The LARG gene expression in the clinorotated cells decreased to 57.5% of that in the stationary control cells. Values were obtained from seven individual experiments in triplicates and are represented as means ± SE ( p
Figure Legend Snippet: Real-time quantitative PCR . The expression of LARG mRNA was measured by real-time quantitative PCR as described in the Methods section. The LARG gene expression in the clinorotated cells decreased to 57.5% of that in the stationary control cells. Values were obtained from seven individual experiments in triplicates and are represented as means ± SE ( p

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

15) Product Images from "Novel insight from transgenic mice into thyroid hormone resistance and the regulation of thyrotropin"

Article Title: Novel insight from transgenic mice into thyroid hormone resistance and the regulation of thyrotropin

Journal: Journal of Clinical Investigation

doi:

Transgenic construct, pituitary expression of the transgene, and impact on basal thyroid hormone concentrations. ( a ) Schematic representation of the transgene. The vector contains 4.6 kb of the mouse glycoprotein subunit α gene cloned upstream of the human TR-β1 cDNA harboring the mutation Δ337T and ligated to a 400-bp fragment containing the SV40 polyadenylation signal. ( b ) RT-PCR scheme for detecting transgene expression and expression of the mutant and wild-type TR transcripts in mouse pituitaries and other tissues. RNA was obtained from pituitary, hypothalamus, liver, kidney, and heart, and analyzed by RT-PCR using primers (denoted a and b ) that are homologous both to the human and mouse TR sequences. These primers amplify 261 bp of the carboxyl-terminus of the human and mouse TR-β gene. A polymorphism in the human gene results in the presence of a Sma 1 restriction site that is not present in the mouse gene. Restriction digestion with this enzyme results in the appearance of 126/137-bp overlapping fragments indicating transgene expression. Gels shown were performed after Sma1 digestion of the RT-PCR products. Note that the smaller transcript appears only in the pituitaries of TG mice but is absent from WT mouse pituitaries and from multiple other tissues, including the hypothalamus in TG mice. Data is shown for line 1 mice; similar results were obtained for line 2 mice, which had 50% lower pituitary transgene expression vs. line 1. ( c ) Basal total T 4 , TSH concentrations, and TSH/T 4 ratios (means ± SEM) in the WT and TG mice from two independent transgenic founder lines (1 and 2). The number of animals used for T 4 determinations are WT: n = 89; line 1: n = 84; and line 2: n = 39, respectively. The number of animals used for TSH determinations are WT: n = 51; line 1: n = 40; and line 2: n = 46. The number of samples on which T 4 and TSH were simultaneously measured for the purpose of calculating the TSH/T 4 ratio are WT: n = 24; line 1: n = 26; and line 2: n = 26. Note the small increase in T 4 concentration in line 1 and the lack of T 4 elevation in line 2, despite large increases in TSH concentration. ** P
Figure Legend Snippet: Transgenic construct, pituitary expression of the transgene, and impact on basal thyroid hormone concentrations. ( a ) Schematic representation of the transgene. The vector contains 4.6 kb of the mouse glycoprotein subunit α gene cloned upstream of the human TR-β1 cDNA harboring the mutation Δ337T and ligated to a 400-bp fragment containing the SV40 polyadenylation signal. ( b ) RT-PCR scheme for detecting transgene expression and expression of the mutant and wild-type TR transcripts in mouse pituitaries and other tissues. RNA was obtained from pituitary, hypothalamus, liver, kidney, and heart, and analyzed by RT-PCR using primers (denoted a and b ) that are homologous both to the human and mouse TR sequences. These primers amplify 261 bp of the carboxyl-terminus of the human and mouse TR-β gene. A polymorphism in the human gene results in the presence of a Sma 1 restriction site that is not present in the mouse gene. Restriction digestion with this enzyme results in the appearance of 126/137-bp overlapping fragments indicating transgene expression. Gels shown were performed after Sma1 digestion of the RT-PCR products. Note that the smaller transcript appears only in the pituitaries of TG mice but is absent from WT mouse pituitaries and from multiple other tissues, including the hypothalamus in TG mice. Data is shown for line 1 mice; similar results were obtained for line 2 mice, which had 50% lower pituitary transgene expression vs. line 1. ( c ) Basal total T 4 , TSH concentrations, and TSH/T 4 ratios (means ± SEM) in the WT and TG mice from two independent transgenic founder lines (1 and 2). The number of animals used for T 4 determinations are WT: n = 89; line 1: n = 84; and line 2: n = 39, respectively. The number of animals used for TSH determinations are WT: n = 51; line 1: n = 40; and line 2: n = 46. The number of samples on which T 4 and TSH were simultaneously measured for the purpose of calculating the TSH/T 4 ratio are WT: n = 24; line 1: n = 26; and line 2: n = 26. Note the small increase in T 4 concentration in line 1 and the lack of T 4 elevation in line 2, despite large increases in TSH concentration. ** P

Techniques Used: Transgenic Assay, Construct, Expressing, Plasmid Preparation, Clone Assay, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Concentration Assay

16) Product Images from "Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3′-side of the transgene"

Article Title: Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3′-side of the transgene

Journal: Oncology Letters

doi: 10.3892/ol.2017.6201

Time course analysis of REIC/Dkk-3 expression levels and associated intracellular signaling. (A) Time course evaluation of REIC/Dkk-3 expression levels from REIC/Dkk-3 cDNA-carrying C-TSC and C-T plasmids was performed in HEK293T cells by western blotting.
Figure Legend Snippet: Time course analysis of REIC/Dkk-3 expression levels and associated intracellular signaling. (A) Time course evaluation of REIC/Dkk-3 expression levels from REIC/Dkk-3 cDNA-carrying C-TSC and C-T plasmids was performed in HEK293T cells by western blotting.

Techniques Used: Expressing, Western Blot

Schematic diagram of the improved gene expression system and assessment of its gene expression. (A) A series of indicated plasmids were constructed on the basis of the promoter-less pIDT-SMART (Km) vector. (B) Expression of REIC/Dkk-3 protein was assessed
Figure Legend Snippet: Schematic diagram of the improved gene expression system and assessment of its gene expression. (A) A series of indicated plasmids were constructed on the basis of the promoter-less pIDT-SMART (Km) vector. (B) Expression of REIC/Dkk-3 protein was assessed

Techniques Used: Expressing, Construct, Plasmid Preparation

Adaptation of the C-T cassette to the conventional Ad-REIC and evaluation of the adapted vector, Ad-C-T-REIC, for gene expression and induction of apoptosis. (A) Expression levels of REIC/Dkk-3 from the indicated adenovirus vectors was determined in a
Figure Legend Snippet: Adaptation of the C-T cassette to the conventional Ad-REIC and evaluation of the adapted vector, Ad-C-T-REIC, for gene expression and induction of apoptosis. (A) Expression levels of REIC/Dkk-3 from the indicated adenovirus vectors was determined in a

Techniques Used: Plasmid Preparation, Expressing

17) Product Images from "Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord"

Article Title: Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord

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

doi:

Tissue distribution of human prepro-UII mRNA. ( A ) Dot blot analysis of prepro-UII mRNA expression in various human tissues. The master blot (CLONTECH) consisted of 50 human-tissue poly(A) RNAs (80–448 ng per dot) normalized by using the RNA expression levels of eight housekeeping genes. Positive controls consisted of human genomic DNA. Negative controls included yeast and Escherichia coli RNA or DNA, as well as human repetitive genomic sequences. The blot was probed with the human prepro-UII cDNA probe and exposed for 2 days onto an X-Omat film ( B ) Northern blot analysis of prepro-UII mRNA expression in the human spinal cord. Spinal cord poly(A) mRNA (2 μg) was hybridized with the human prepro-UII cDNA probe. The molecular weight was determined by using RNA markers. ( C ) X-ray autoradiographs showing the distribution of prepro-UII mRNA in the human spinal cord. Coronal sections were hybridized with the antisense ( Top ) or sense ( Bottom ) prepro-UII riboprobe and exposed for 10 days onto x-ray film.
Figure Legend Snippet: Tissue distribution of human prepro-UII mRNA. ( A ) Dot blot analysis of prepro-UII mRNA expression in various human tissues. The master blot (CLONTECH) consisted of 50 human-tissue poly(A) RNAs (80–448 ng per dot) normalized by using the RNA expression levels of eight housekeeping genes. Positive controls consisted of human genomic DNA. Negative controls included yeast and Escherichia coli RNA or DNA, as well as human repetitive genomic sequences. The blot was probed with the human prepro-UII cDNA probe and exposed for 2 days onto an X-Omat film ( B ) Northern blot analysis of prepro-UII mRNA expression in the human spinal cord. Spinal cord poly(A) mRNA (2 μg) was hybridized with the human prepro-UII cDNA probe. The molecular weight was determined by using RNA markers. ( C ) X-ray autoradiographs showing the distribution of prepro-UII mRNA in the human spinal cord. Coronal sections were hybridized with the antisense ( Top ) or sense ( Bottom ) prepro-UII riboprobe and exposed for 10 days onto x-ray film.

Techniques Used: Dot Blot, Expressing, RNA Expression, Genomic Sequencing, Northern Blot, Molecular Weight

Tissue distribution of frog prepro-UII mRNA. ( A ) Northern blot analysis of prepro-UII mRNA expression in the frog central nervous system. Total RNA (30 μg) was electrophoresed on a formaldehyde–agarose gel, transferred onto a nylon membrane and probed with the frog prepro-UII cDNA probe. ( B ) Analysis of prepro-UII mRNA distribution in various frog tissues by RT-PCR. Total RNA (5 μg) was reverse transcribed, amplified with prepro-UII specific primers, electrophoresed on an agarose gel and stained with ethidium bromide ( Upper ). The gel was then blotted and hybridized with an internal prepro-UII primer ( Lower ). The control lane contained no DNA. The sizes of the PCR products were evaluated by using a DNA mass ladder (M).
Figure Legend Snippet: Tissue distribution of frog prepro-UII mRNA. ( A ) Northern blot analysis of prepro-UII mRNA expression in the frog central nervous system. Total RNA (30 μg) was electrophoresed on a formaldehyde–agarose gel, transferred onto a nylon membrane and probed with the frog prepro-UII cDNA probe. ( B ) Analysis of prepro-UII mRNA distribution in various frog tissues by RT-PCR. Total RNA (5 μg) was reverse transcribed, amplified with prepro-UII specific primers, electrophoresed on an agarose gel and stained with ethidium bromide ( Upper ). The gel was then blotted and hybridized with an internal prepro-UII primer ( Lower ). The control lane contained no DNA. The sizes of the PCR products were evaluated by using a DNA mass ladder (M).

Techniques Used: Northern Blot, Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Staining, Polymerase Chain Reaction

18) Product Images from "LncRNA HOTAIR promotes cell migration and invasion by regulating MKL1 via inhibition miR206 expression in HeLa cells"

Article Title: LncRNA HOTAIR promotes cell migration and invasion by regulating MKL1 via inhibition miR206 expression in HeLa cells

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/s12964-018-0216-3

MKL1 activated HOTAIR transcription by binding the CArG box in promoter region. a Diagram of the CArG box in HOTAIR promoter region. b Cells were transfected with luciferase vectors containing the promoter region of HOTAIR, along with MKL1 expression vector. Luciferase activity was measured at 48 h after transfection. HOTAIR promoter activity was decreased after transfecting with plasmids, which mutated or deleted CArG box in the promoter region of HOTAIR, compared with the control. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P
Figure Legend Snippet: MKL1 activated HOTAIR transcription by binding the CArG box in promoter region. a Diagram of the CArG box in HOTAIR promoter region. b Cells were transfected with luciferase vectors containing the promoter region of HOTAIR, along with MKL1 expression vector. Luciferase activity was measured at 48 h after transfection. HOTAIR promoter activity was decreased after transfecting with plasmids, which mutated or deleted CArG box in the promoter region of HOTAIR, compared with the control. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P

Techniques Used: Binding Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay

miR-206 degraded MKL1 by targeting the specific sites. a HeLa cells were transfected with miR-206 mimics or NC for 48 h. The abundance of miR-206 expression was determined by qRT-PCR. The expression level of miR-206 was normalized to U6. b Western blots analysis of the MKL1 protein expression at 48 h after transfected with miR-206 mimics or NC. GAPDH was used as an internal control. c qRT-PCR was used to measure the efficiency of miR-206 inhibition after transfecting miR-206 inhibitor at 48 h. The expression level of miR-206 was normalized to U6. d Western blots analysis of the MKL1 protein expression at 48 h after transfected with miR-206 inhibitor or NC. GAPDH was used as an internal control. e Diagram of MKL1–3’UTR containing reporter constructs. The seed sequence of miR-206 was underlined. Mutation contains 4-base-mutation at the miR-206 targeting region, abolishing its binding. f. In luciferase assays using HeLa cells, transfection of miR-206 and MKL1–3’UTR-mut increased the luciferase activities compared with transfection of miR-206 and MKL1–3’UTR-WT. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P
Figure Legend Snippet: miR-206 degraded MKL1 by targeting the specific sites. a HeLa cells were transfected with miR-206 mimics or NC for 48 h. The abundance of miR-206 expression was determined by qRT-PCR. The expression level of miR-206 was normalized to U6. b Western blots analysis of the MKL1 protein expression at 48 h after transfected with miR-206 mimics or NC. GAPDH was used as an internal control. c qRT-PCR was used to measure the efficiency of miR-206 inhibition after transfecting miR-206 inhibitor at 48 h. The expression level of miR-206 was normalized to U6. d Western blots analysis of the MKL1 protein expression at 48 h after transfected with miR-206 inhibitor or NC. GAPDH was used as an internal control. e Diagram of MKL1–3’UTR containing reporter constructs. The seed sequence of miR-206 was underlined. Mutation contains 4-base-mutation at the miR-206 targeting region, abolishing its binding. f. In luciferase assays using HeLa cells, transfection of miR-206 and MKL1–3’UTR-mut increased the luciferase activities compared with transfection of miR-206 and MKL1–3’UTR-WT. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P

Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Inhibition, Construct, Sequencing, Mutagenesis, Binding Assay, Luciferase

Inhibition of HOTAIR affects the location of MKL1 a. Representative images showing the distribution of MKL1 in HeLa cells after HOTAIR knockdown under confocal microscopy. b Western blotting analysis the expression of MKL1 in cytoplasm and nucleus after HOTAIR inhibition. c Representative images showing the distribution of MKL1 in HeLa cells after MKL1 knockdown under confocal microscopy. d Western blotting analysis the expression of MKL1 in cytoplasm and nucleus after transfecting siRNA targeted against MKL1. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P
Figure Legend Snippet: Inhibition of HOTAIR affects the location of MKL1 a. Representative images showing the distribution of MKL1 in HeLa cells after HOTAIR knockdown under confocal microscopy. b Western blotting analysis the expression of MKL1 in cytoplasm and nucleus after HOTAIR inhibition. c Representative images showing the distribution of MKL1 in HeLa cells after MKL1 knockdown under confocal microscopy. d Western blotting analysis the expression of MKL1 in cytoplasm and nucleus after transfecting siRNA targeted against MKL1. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P

Techniques Used: Inhibition, Confocal Microscopy, Western Blot, Expressing

Western blotting determine MKL1 expression. a HeLa cells were transfected with siHOTAIR or siNC for 48 h. The efficiency of HOTAIR knockdown was determined by qRT-PCR. The expression level of HOTAIR was normalized to GAPDH. b Western blots analysis of the MKL1 protein expression at 48 h after transfected with siHOTAIR (siHOTAIR-I and siHOTAIR-II) or siNC. GAPDH was used as an internal control. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P
Figure Legend Snippet: Western blotting determine MKL1 expression. a HeLa cells were transfected with siHOTAIR or siNC for 48 h. The efficiency of HOTAIR knockdown was determined by qRT-PCR. The expression level of HOTAIR was normalized to GAPDH. b Western blots analysis of the MKL1 protein expression at 48 h after transfected with siHOTAIR (siHOTAIR-I and siHOTAIR-II) or siNC. GAPDH was used as an internal control. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P

Techniques Used: Western Blot, Expressing, Transfection, Quantitative RT-PCR

Overexpression MKL1 promoted cell migration and invasion in HeLa cells. a HeLa cells were transfected with pMKL1 or pCDNA3.1. The MKL1 expression level was determined by western blot at 24 h and 48 h after transfection. pCDNA3.1 serves as the negative control and GAPDH serves as the loading control. b The effect of MKL1 overexpression on cell migration was determined by wound healing assay. c Quantification of the wound healing assay. d The effect of MKL1 overexpression on cell invasion was determined in a Boyden chamber assay. e The number of cells on the underside of the filter was determined and significantly ( P
Figure Legend Snippet: Overexpression MKL1 promoted cell migration and invasion in HeLa cells. a HeLa cells were transfected with pMKL1 or pCDNA3.1. The MKL1 expression level was determined by western blot at 24 h and 48 h after transfection. pCDNA3.1 serves as the negative control and GAPDH serves as the loading control. b The effect of MKL1 overexpression on cell migration was determined by wound healing assay. c Quantification of the wound healing assay. d The effect of MKL1 overexpression on cell invasion was determined in a Boyden chamber assay. e The number of cells on the underside of the filter was determined and significantly ( P

Techniques Used: Over Expression, Migration, Transfection, Expressing, Western Blot, Negative Control, Wound Healing Assay, Boyden Chamber Assay

High level of MKL1 significantly correlate with HOTAIR expression in Cervical cancer patient. a Hematoxylin and eosin (HE)-stained cervical cancer tissues and adjacent normal tissues. b HOTAIR stained by in situ hybridization; high level of HOTAIR expression in cervical cancer tissues compared with adjacent normal tissues. c The expression of MKL1 was detected in cervical cancer tissues and adjacent normal tissues by IHC. The MKL1 expression was enhanced in cervical cancer tissues compared with adjacent normal tissues. The images were acquired by Pannoramic MIDI with a 20 × microscope objective. d The correlation between the expression of HOTAIR and MKL1 in cervical cancer tissues from 31 patients was shown. Red represented the RNA expression of HOTAIR. And Green represented MKL1 protein expression level
Figure Legend Snippet: High level of MKL1 significantly correlate with HOTAIR expression in Cervical cancer patient. a Hematoxylin and eosin (HE)-stained cervical cancer tissues and adjacent normal tissues. b HOTAIR stained by in situ hybridization; high level of HOTAIR expression in cervical cancer tissues compared with adjacent normal tissues. c The expression of MKL1 was detected in cervical cancer tissues and adjacent normal tissues by IHC. The MKL1 expression was enhanced in cervical cancer tissues compared with adjacent normal tissues. The images were acquired by Pannoramic MIDI with a 20 × microscope objective. d The correlation between the expression of HOTAIR and MKL1 in cervical cancer tissues from 31 patients was shown. Red represented the RNA expression of HOTAIR. And Green represented MKL1 protein expression level

Techniques Used: Expressing, Staining, In Situ Hybridization, Immunohistochemistry, Microscopy, RNA Expression

a Western blots analysis of the MKL1 protein expression at 48 h after transfected with siMKL1 (siMKL1-I, siMKL1-II and siMKL1-III) or siNC. GAPDH was used as an internal control. b The effect of knockdown HOTAIR or/and MKL1 on cell migration was determined by wound healing assay. c Quantification of the wound healing assay. d, e, f The effect of HOTAIR or/and MKL1 inhibition on cell invasion was determined in a Boyden chamber assay. And the number of cells on the underside of the filter was determined and significantly ( P
Figure Legend Snippet: a Western blots analysis of the MKL1 protein expression at 48 h after transfected with siMKL1 (siMKL1-I, siMKL1-II and siMKL1-III) or siNC. GAPDH was used as an internal control. b The effect of knockdown HOTAIR or/and MKL1 on cell migration was determined by wound healing assay. c Quantification of the wound healing assay. d, e, f The effect of HOTAIR or/and MKL1 inhibition on cell invasion was determined in a Boyden chamber assay. And the number of cells on the underside of the filter was determined and significantly ( P

Techniques Used: Western Blot, Expressing, Transfection, Migration, Wound Healing Assay, Inhibition, Boyden Chamber Assay

19) Product Images from "Insights from lncRNAs Profiling of MIN6 Beta Cells Undergoing Inflammation"

Article Title: Insights from lncRNAs Profiling of MIN6 Beta Cells Undergoing Inflammation

Journal: Mediators of Inflammation

doi: 10.1155/2016/9275106

Comparison between microarray data and qRT-PCR result for lncRNAs. The validation results indicated that the microarray data correlated well with the qPCR results.
Figure Legend Snippet: Comparison between microarray data and qRT-PCR result for lncRNAs. The validation results indicated that the microarray data correlated well with the qPCR results.

Techniques Used: Microarray, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

20) Product Images from "MiR-199a/b-5p Inhibits Lymphangiogenesis by Targeting Discoidin Domain Receptor 1 in Corneal Injury"

Article Title: MiR-199a/b-5p Inhibits Lymphangiogenesis by Targeting Discoidin Domain Receptor 1 in Corneal Injury

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.2163

Effect of miR-199a/b-5p inhibitors on HDLEC tube formation (A) Effect of miRNA inhibitors on the level of miR-199a/b-5p. HDLECs were transfected with 20 nM miR-199a/b-5p inhibitors or the negative control inhibitor. After 48 h, cells were harvested for qRT-PCR analysis; three independent experiments were performed, and relative miR-199a/b-5p expression was calculated according to the comparative Ct method, using U6 as an internal control ( n = 3). Error bars indicate SD. (B) Western blot analysis to detect DDR1 expression 48 h after transfection with miR-199a/b-5p inhibitors. Three sets of independent experiments were performed and representative results are shown (upper panel). Anti-β-actin antibody (1:1000) was used to confirm comparable loading. Band densities were quantified using Fujifilm Multi Gauge software version 3.0, and relative DDR1 expression levels are plotted (lower panel). Error bars indicate SD ( n = 3). (C) At 48 h post-transfection, cells (10 5 cells/well) were plated in an ECMatrix-coated eight-well chamber in MV2 medium, containing 10 ng/ml bFGF, 5 ng/ml EGF, and 20 ng/ml IGF-1. To detect the tube formation more clearly, the cells were stained in a media containing 5 μM calcein AM for 1 h. Capillary-like structures within the Matrigel layer formed by HDLECs were photographed after 4–6 h at 50× magnification. Three independent experiments were performed for each setting and representative results are shown at the left panels. Tube formation was quantified using ImageJ software, and the mean +/− SD values are plotted on the right panels ( n = 3).
Figure Legend Snippet: Effect of miR-199a/b-5p inhibitors on HDLEC tube formation (A) Effect of miRNA inhibitors on the level of miR-199a/b-5p. HDLECs were transfected with 20 nM miR-199a/b-5p inhibitors or the negative control inhibitor. After 48 h, cells were harvested for qRT-PCR analysis; three independent experiments were performed, and relative miR-199a/b-5p expression was calculated according to the comparative Ct method, using U6 as an internal control ( n = 3). Error bars indicate SD. (B) Western blot analysis to detect DDR1 expression 48 h after transfection with miR-199a/b-5p inhibitors. Three sets of independent experiments were performed and representative results are shown (upper panel). Anti-β-actin antibody (1:1000) was used to confirm comparable loading. Band densities were quantified using Fujifilm Multi Gauge software version 3.0, and relative DDR1 expression levels are plotted (lower panel). Error bars indicate SD ( n = 3). (C) At 48 h post-transfection, cells (10 5 cells/well) were plated in an ECMatrix-coated eight-well chamber in MV2 medium, containing 10 ng/ml bFGF, 5 ng/ml EGF, and 20 ng/ml IGF-1. To detect the tube formation more clearly, the cells were stained in a media containing 5 μM calcein AM for 1 h. Capillary-like structures within the Matrigel layer formed by HDLECs were photographed after 4–6 h at 50× magnification. Three independent experiments were performed for each setting and representative results are shown at the left panels. Tube formation was quantified using ImageJ software, and the mean +/− SD values are plotted on the right panels ( n = 3).

Techniques Used: Transfection, Negative Control, Quantitative RT-PCR, Expressing, Western Blot, Software, Staining

Effects of miR-199a/b-5p mimics on expression of DDR1 mRNA and protein (A) Sequence of miR-199a/b-5p and the mutant forms of miR-199a/b-5p (miR-199a/b-5pm); mutants have nucleotide substitutions at two to four sites within the miRNA. (B) Reduction in DDR1 mRNA levels by miR-199a/b-5p. HDLECs were transfected with 20 nM miR-199a/b-5p mimics or the scrambled control, and levels of DDR1 mRNA were measured by qRT-PCR. Relative gene expression was calculated according to the comparative Ct method, using GAPDH as an internal control ( n = 3). Error bars indicate standard deviation (SD). (C) Effects of miR-199a/b-5p on DDR1 protein expression. HDLECs were transfected with 20 nM miR-199a/b-5p mimics or the scrambled control. To confirm the sequence-specific function of miR-199a/b-5p, miR-199a/b-5p mutants (miR-199a/b-5pm) were also transfected. Cell lysates were examined by Western blot analysis with an anti-DDR1 polyclonal antibody (1:500) at 48 h post-transfection; an anti-β-actin antibody (1:1000) was used to normalize protein loading. Three sets of independent experiments were performed, and representative results are shown in the lower panel; asterisk ( * ) indicates nonspecific band. The density of each protein band was quantified using Fujifilm Multi Gauge software version 3.0 and expressed as a ratio to the density of the band from the scrambled control. Upper panel shows mean ± SD values of the three independent experiments.
Figure Legend Snippet: Effects of miR-199a/b-5p mimics on expression of DDR1 mRNA and protein (A) Sequence of miR-199a/b-5p and the mutant forms of miR-199a/b-5p (miR-199a/b-5pm); mutants have nucleotide substitutions at two to four sites within the miRNA. (B) Reduction in DDR1 mRNA levels by miR-199a/b-5p. HDLECs were transfected with 20 nM miR-199a/b-5p mimics or the scrambled control, and levels of DDR1 mRNA were measured by qRT-PCR. Relative gene expression was calculated according to the comparative Ct method, using GAPDH as an internal control ( n = 3). Error bars indicate standard deviation (SD). (C) Effects of miR-199a/b-5p on DDR1 protein expression. HDLECs were transfected with 20 nM miR-199a/b-5p mimics or the scrambled control. To confirm the sequence-specific function of miR-199a/b-5p, miR-199a/b-5p mutants (miR-199a/b-5pm) were also transfected. Cell lysates were examined by Western blot analysis with an anti-DDR1 polyclonal antibody (1:500) at 48 h post-transfection; an anti-β-actin antibody (1:1000) was used to normalize protein loading. Three sets of independent experiments were performed, and representative results are shown in the lower panel; asterisk ( * ) indicates nonspecific band. The density of each protein band was quantified using Fujifilm Multi Gauge software version 3.0 and expressed as a ratio to the density of the band from the scrambled control. Upper panel shows mean ± SD values of the three independent experiments.

Techniques Used: Expressing, Sequencing, Mutagenesis, Transfection, Quantitative RT-PCR, Standard Deviation, Western Blot, Software

21) Product Images from "Isolation and expression of a Pax-6 gene in the regenerating and intact Planarian Dugesia(G)tigrina"

Article Title: Isolation and expression of a Pax-6 gene in the regenerating and intact Planarian Dugesia(G)tigrina

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

doi:

DtPax-6 expression in Dugesia(G)tigrina . ( A ) Northern blot: 10 μg of poly(A) + from intact adults (lane 1) and 7-day-regenerating (lane 2) planarians hybridized at high stringency conditions with 0.55-kb DtPax-6 3′ cDNA fragment. A single band of 2.1 kb was observed in both lanes. Blots were rehybridized with a Drosophila ) to control for levels of RNA loaded. The transcript size and the positions of 23S and 16S RNAs are indicated. ( B ) Adult planarian DtPax-6 ) regions deduced by RT-PCR amplification using specific oligonucleotides from exons 3 and 4 from the DtPax-6 cDNA sequence (positions 1226–1250 and 1363–1387, respectively) that produce a fragment of 162 nt. As an internal control, we used in the same amplification two new sets of specific oligonucleotides from the Dth-2 ), respectively] that produce a control fragment of 465 nt. Lane M, 100-bp ladder (Pharmacia) molecular size marker. Lane C, non-retro-transcribed planarian total RNA as a control.
Figure Legend Snippet: DtPax-6 expression in Dugesia(G)tigrina . ( A ) Northern blot: 10 μg of poly(A) + from intact adults (lane 1) and 7-day-regenerating (lane 2) planarians hybridized at high stringency conditions with 0.55-kb DtPax-6 3′ cDNA fragment. A single band of 2.1 kb was observed in both lanes. Blots were rehybridized with a Drosophila ) to control for levels of RNA loaded. The transcript size and the positions of 23S and 16S RNAs are indicated. ( B ) Adult planarian DtPax-6 ) regions deduced by RT-PCR amplification using specific oligonucleotides from exons 3 and 4 from the DtPax-6 cDNA sequence (positions 1226–1250 and 1363–1387, respectively) that produce a fragment of 162 nt. As an internal control, we used in the same amplification two new sets of specific oligonucleotides from the Dth-2 ), respectively] that produce a control fragment of 465 nt. Lane M, 100-bp ladder (Pharmacia) molecular size marker. Lane C, non-retro-transcribed planarian total RNA as a control.

Techniques Used: Expressing, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Marker

22) Product Images from "Establishment and characterization of an immortalized human hepatocyte line for the development of bioartificial liver system"

Article Title: Establishment and characterization of an immortalized human hepatocyte line for the development of bioartificial liver system

Journal: Cytotechnology

doi: 10.1007/s10616-017-0167-3

Gene expression profiling of NHBL2: A the heatmap image of NHBL2 and C3A cell lines gene data. B The corrmatrix of NHBL2 and C3A cell lines gene data, the correlation of all samples was very high, over 0.98. C The expression of most genes was at the same level between NHBL2 and C3A cells. D The results of microarray were further confirmed by q-PCR assay and GAPDH was used as an internal reference
Figure Legend Snippet: Gene expression profiling of NHBL2: A the heatmap image of NHBL2 and C3A cell lines gene data. B The corrmatrix of NHBL2 and C3A cell lines gene data, the correlation of all samples was very high, over 0.98. C The expression of most genes was at the same level between NHBL2 and C3A cells. D The results of microarray were further confirmed by q-PCR assay and GAPDH was used as an internal reference

Techniques Used: Expressing, Microarray, Polymerase Chain Reaction

A PAS staining of C3A (a, 400×) and NHBL2 (b, 400×). B Detection of ALB and BUN from culture supernatants of NHBL2 cell line (* P
Figure Legend Snippet: A PAS staining of C3A (a, 400×) and NHBL2 (b, 400×). B Detection of ALB and BUN from culture supernatants of NHBL2 cell line (* P

Techniques Used: Staining

23) Product Images from "Indole-3-Acetic Acid Biosynthesis Pathways in the Plant-Beneficial Bacterium Arthrobacter pascens ZZ21"

Article Title: Indole-3-Acetic Acid Biosynthesis Pathways in the Plant-Beneficial Bacterium Arthrobacter pascens ZZ21

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19020443

IAA production and genes transcript levels in ZZ21 in response to tryptophan. ( a ) IAA production in A. pascens ZZ21 cultured for 48 h with or without 200 mg·L −1 l -tryptophan in Luria-Bertani (LB) medium. ( b ) Relative expression of genes in ZZ21 cultured in the absence or presence of tryptophan (Trp). Gene transcript levels were evaluated by qRT-PCR. ZZ21 was grown in LB medium with or without tryptophan for 48 h. The 16sRNA gene of ZZ21 was used as an internal reference. Error bars represent standard errors of three biological replicates. One-way analysis of variance (ANOVA) was performed. * p
Figure Legend Snippet: IAA production and genes transcript levels in ZZ21 in response to tryptophan. ( a ) IAA production in A. pascens ZZ21 cultured for 48 h with or without 200 mg·L −1 l -tryptophan in Luria-Bertani (LB) medium. ( b ) Relative expression of genes in ZZ21 cultured in the absence or presence of tryptophan (Trp). Gene transcript levels were evaluated by qRT-PCR. ZZ21 was grown in LB medium with or without tryptophan for 48 h. The 16sRNA gene of ZZ21 was used as an internal reference. Error bars represent standard errors of three biological replicates. One-way analysis of variance (ANOVA) was performed. * p

Techniques Used: Cell Culture, Expressing, Quantitative RT-PCR

24) Product Images from "Melatonin receptor heterodimerization in a photoreceptor-like cell line endogenously expressing melatonin receptors"

Article Title: Melatonin receptor heterodimerization in a photoreceptor-like cell line endogenously expressing melatonin receptors

Journal: Molecular Vision

doi:

Genetic ablation of MT 2 with CRISPR/Cas9 system does not affect MT 1 mRNA or protein. Top panels: cDNA from 661W and 661W-MT 2 −/− cells was analyzed with reverse transcriptase PCR (RT–PCR) to detect MT 1 (left) and MT 2 (right) expression. Middle panels: Immunofluorescence was performed in 661W and 661W-melatonin receptor type 2 (MT 2 ) −/− cells to detect MT 1 (left) and MT 2 (right) proteins. The positive signal of each receptor is shown in green, and nuclei are counterstained in red. Lower panel: The presence or absence of MT 1 /MT 2 heterodimer in 661W and 661W-MT 2 −/− cells was assessed with proximity ligation assay (PLA; positive signal in red), and nuclei were counterstained in green. MT 1 expression (transcript and protein) was detected in 661W and 661W-MT 2 −/− cells, whereas MT 2 expression (transcript and protein) was detected only in 661W cells. Accordingly, MT 1/2h was found only in 661W cells. Scale bars = 100 µm.
Figure Legend Snippet: Genetic ablation of MT 2 with CRISPR/Cas9 system does not affect MT 1 mRNA or protein. Top panels: cDNA from 661W and 661W-MT 2 −/− cells was analyzed with reverse transcriptase PCR (RT–PCR) to detect MT 1 (left) and MT 2 (right) expression. Middle panels: Immunofluorescence was performed in 661W and 661W-melatonin receptor type 2 (MT 2 ) −/− cells to detect MT 1 (left) and MT 2 (right) proteins. The positive signal of each receptor is shown in green, and nuclei are counterstained in red. Lower panel: The presence or absence of MT 1 /MT 2 heterodimer in 661W and 661W-MT 2 −/− cells was assessed with proximity ligation assay (PLA; positive signal in red), and nuclei were counterstained in green. MT 1 expression (transcript and protein) was detected in 661W and 661W-MT 2 −/− cells, whereas MT 2 expression (transcript and protein) was detected only in 661W cells. Accordingly, MT 1/2h was found only in 661W cells. Scale bars = 100 µm.

Techniques Used: CRISPR, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence, Proximity Ligation Assay

25) Product Images from "Foot-and-mouth disease virus O/ME-SA/Ind 2001 lineage outbreak in vaccinated Holstein Friesian cattle in Saudi Arabia in 2016"

Article Title: Foot-and-mouth disease virus O/ME-SA/Ind 2001 lineage outbreak in vaccinated Holstein Friesian cattle in Saudi Arabia in 2016

Journal: The Veterinary Quarterly

doi: 10.1080/01652176.2018.1539568

Agarose gel electrophoresis of some RT-PCR-tested nasal swabs from cattle for FMDV in 2016 in Eastern Saudi Arabia. Some RT-PCR results of selected specimens collected from the nasal swabs of FMDV-infected animals. Lane (M) DNA marker, (100 bp); lane (1) empty well; lanes (2–13) purified PCR products of the partial FMDV VP-1 gene. The positive amplicons are 641 bp in length.
Figure Legend Snippet: Agarose gel electrophoresis of some RT-PCR-tested nasal swabs from cattle for FMDV in 2016 in Eastern Saudi Arabia. Some RT-PCR results of selected specimens collected from the nasal swabs of FMDV-infected animals. Lane (M) DNA marker, (100 bp); lane (1) empty well; lanes (2–13) purified PCR products of the partial FMDV VP-1 gene. The positive amplicons are 641 bp in length.

Techniques Used: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Infection, Marker, Purification, Polymerase Chain Reaction

26) Product Images from "CircCDYL Serves as a New Biomarker in Mantle Cell Lymphoma and Promotes Cell Proliferation"

Article Title: CircCDYL Serves as a New Biomarker in Mantle Cell Lymphoma and Promotes Cell Proliferation

Journal: Cancer Management and Research

doi: 10.2147/CMAR.S232075

Circ-CDYL was upregulated in MCL patients. ( A ) qRT-PCR analysis showed that Circ-CDYL was upregulated in MCL patients (n = 18) compared with normal controls (n = 17). ( B ) ROC curve showed that Circ-CDYL expression could serve as a diagnostic biomarker in MCL. ( C ) Kaplan–Meier OS curves for MCL patients stratified according to circ-CDYL expression. ***p
Figure Legend Snippet: Circ-CDYL was upregulated in MCL patients. ( A ) qRT-PCR analysis showed that Circ-CDYL was upregulated in MCL patients (n = 18) compared with normal controls (n = 17). ( B ) ROC curve showed that Circ-CDYL expression could serve as a diagnostic biomarker in MCL. ( C ) Kaplan–Meier OS curves for MCL patients stratified according to circ-CDYL expression. ***p

Techniques Used: Quantitative RT-PCR, Expressing, Diagnostic Assay, Biomarker Assay

27) Product Images from "Comparative Transcriptomics Reveals Differential Gene Expression Related to Colletotrichum gloeosporioides Resistance in the Octoploid Strawberry"

Article Title: Comparative Transcriptomics Reveals Differential Gene Expression Related to Colletotrichum gloeosporioides Resistance in the Octoploid Strawberry

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.00779

Relative expression values according to the qRT-PCR analysis of the up-regulated strawberry genes in the plant–pathogen interaction pathways between mock and infected leaves at 72 h of ‘Yanli’ (A) and ‘Benihoppe’ (B) . The qPCR results were generally in accordance with the gene expression profiles in the transcriptome data. ∗ p
Figure Legend Snippet: Relative expression values according to the qRT-PCR analysis of the up-regulated strawberry genes in the plant–pathogen interaction pathways between mock and infected leaves at 72 h of ‘Yanli’ (A) and ‘Benihoppe’ (B) . The qPCR results were generally in accordance with the gene expression profiles in the transcriptome data. ∗ p

Techniques Used: Expressing, Quantitative RT-PCR, Infection, Real-time Polymerase Chain Reaction

28) Product Images from "Cloning and Functional Characterization of Human SMCT2 (SLC5A12) and Expression Pattern of the Transporter in Kidney"

Article Title: Cloning and Functional Characterization of Human SMCT2 (SLC5A12) and Expression Pattern of the Transporter in Kidney

Journal: Biochimica et biophysica acta

doi: 10.1016/j.bbamem.2007.06.031

Dose-response relationship for the inhibition of human SMCT2-mediated [ 14 C]nicotinate uptake by unlabeled nicotinate, lactate, and butyrate (A) and inhibition of human SMCT2-mediated nicotinate uptake by NSAIDs (B). Human SMCT2 cDNA was expressed in HRPE cells. Cells transfected with pcDNA3.1 vector served to determine endogenous transport. Uptake of [ 14 C]nicotinate (30 μM) was measured in Na + -containing buffer either in the presence of increasing concentrations of unlabelled nicotinate, L-lactate, or butyrate (A) or various NSAIDs (200 μM) (B). Uptake measured in cells transfected with vector alone was subtracted from corresponding uptake measured in cells transfected with cDNA to calculate cDNA-specific uptake. Results are expressed as percentage of control uptake (100 %) measured in the absence of inhibitors. The degree of statistical significance for nicotinate uptake measured in the presence of NSAIDs compared to nicotinate uptake measured in the absence of various NSAIDs in hSMCT2 cDNA-transfected cells is indicated by * P
Figure Legend Snippet: Dose-response relationship for the inhibition of human SMCT2-mediated [ 14 C]nicotinate uptake by unlabeled nicotinate, lactate, and butyrate (A) and inhibition of human SMCT2-mediated nicotinate uptake by NSAIDs (B). Human SMCT2 cDNA was expressed in HRPE cells. Cells transfected with pcDNA3.1 vector served to determine endogenous transport. Uptake of [ 14 C]nicotinate (30 μM) was measured in Na + -containing buffer either in the presence of increasing concentrations of unlabelled nicotinate, L-lactate, or butyrate (A) or various NSAIDs (200 μM) (B). Uptake measured in cells transfected with vector alone was subtracted from corresponding uptake measured in cells transfected with cDNA to calculate cDNA-specific uptake. Results are expressed as percentage of control uptake (100 %) measured in the absence of inhibitors. The degree of statistical significance for nicotinate uptake measured in the presence of NSAIDs compared to nicotinate uptake measured in the absence of various NSAIDs in hSMCT2 cDNA-transfected cells is indicated by * P

Techniques Used: Inhibition, Transfection, Plasmid Preparation

Functional expression of human SMCT2 (SLC5A12) in HRPE cells. HRPE cells were transfected with either pcDNA3.1 vector alone (open bars) or human SMCT2 cDNA (closed bars). ( A, B ) Uptake of [ 14 C]lactate (500 μM), [ 14 C]pyruvate (500 μM), and [ 14 C]nicotinate (30 μM) was measured in the presence of Na + . ( C ) Uptake of [ 14 C]nicotinate was measured either in control buffer (25 mM Hepes/Tris, pH 7.5, 5.4 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgSO 4 , 5 mM glucose, and 140 mM NaCl) or in buffer in which NaCl was replaced with 140 mM of KCl or LiCl. Where indicated, uptake measured in cDNA-transfected cells was significantly different from the corresponding uptake measured in vector-transfected cells (* P
Figure Legend Snippet: Functional expression of human SMCT2 (SLC5A12) in HRPE cells. HRPE cells were transfected with either pcDNA3.1 vector alone (open bars) or human SMCT2 cDNA (closed bars). ( A, B ) Uptake of [ 14 C]lactate (500 μM), [ 14 C]pyruvate (500 μM), and [ 14 C]nicotinate (30 μM) was measured in the presence of Na + . ( C ) Uptake of [ 14 C]nicotinate was measured either in control buffer (25 mM Hepes/Tris, pH 7.5, 5.4 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgSO 4 , 5 mM glucose, and 140 mM NaCl) or in buffer in which NaCl was replaced with 140 mM of KCl or LiCl. Where indicated, uptake measured in cDNA-transfected cells was significantly different from the corresponding uptake measured in vector-transfected cells (* P

Techniques Used: Functional Assay, Expressing, Transfection, Plasmid Preparation

29) Product Images from "The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli"

Article Title: The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli

Journal: Scientific Reports

doi: 10.1038/s41598-019-53883-y

Expression levels of Eno, Rng, Rnc, and cis -antisense RNA depending on oxygen availability. (a) Schematic representation of the aerobic–anaerobic–aerobic alternating experiment. (b) Expression profiles of Eno, Rng, and Rnc depending on oxygen availability. WT MG1655 cells were cultured to each time point under aerobic or anaerobic conditions at 37 °C and then harvested for western blot analysis of Eno, Rng, and Rnc using protein-specific polyclonal antibodies. Blue, red, and yellow bars indicate the relative expression levels of Eno, Rng, and Rnc, respectively. The expression levels of Eno, Rng, and Rnc were compared by setting those of t0 to 1. Significant differences are indicated with different letters (one-way ANOVA followed by the Student–Newman–Keuls test; Small English letters indicate the difference from expression levels of Eno; Large English letters indicate the difference from expression levels of Rng; Greek symbols indicate the difference from expression levels of Rnc). (c) Analysis of cis -antisense RNA and eno mRNA expression in WT MG1655 cells using RT-PCR. The cDNA was synthesised from the total RNA extracted at each time point using the primers designed to bind cis -antisense RNA and eno mRNA. The PCR products were resolved in an 1.5% agarose gel. Significant differences are indicated with different letters (one-way ANOVA followed by the Student–Newman–Keuls test; Small English letters indicate the difference from expression levels of cis -antisense RNA; Large English letters indicate the difference of expression levels of eno mRNA). (d) Effect of Eno expression levels on the growth of W3110 PBAD eno cells. Cultures of W3110 PBAD eno cells were grown in LB medium containing 0.2% glucose under anaerobic conditions to the early log phase (OD 600 = 0.05) and different concentrations of arabinose (0%, 0.01%, or 0.1%) were added to induce Eno synthesis. As a control, WT W3110 cells were grown in the same way described above and 0.1% arabinose was added. Cultures were further grown (5 h after induction) and were monitored by measuring the OD 600 . Cultures were harvested for western blot analysis of Eno using protein-specific polyclonal antibodies. The grey and blue bars indicate the OD 600 values and the relative expression levels of Eno, respectively. The expression levels of Eno were compared by setting those of W3110 to 1. N.S., not significant. For (b,d) , the S1 protein was used as an internal standard to evaluate the amount of cell extract in each lane. For (c) , the rnpB mRNA was used as an internal standard to evaluate the amount of cell extract in each lane. For (b – d) , the data are presented as means ± s. e. m. of at least three independent experiments.
Figure Legend Snippet: Expression levels of Eno, Rng, Rnc, and cis -antisense RNA depending on oxygen availability. (a) Schematic representation of the aerobic–anaerobic–aerobic alternating experiment. (b) Expression profiles of Eno, Rng, and Rnc depending on oxygen availability. WT MG1655 cells were cultured to each time point under aerobic or anaerobic conditions at 37 °C and then harvested for western blot analysis of Eno, Rng, and Rnc using protein-specific polyclonal antibodies. Blue, red, and yellow bars indicate the relative expression levels of Eno, Rng, and Rnc, respectively. The expression levels of Eno, Rng, and Rnc were compared by setting those of t0 to 1. Significant differences are indicated with different letters (one-way ANOVA followed by the Student–Newman–Keuls test; Small English letters indicate the difference from expression levels of Eno; Large English letters indicate the difference from expression levels of Rng; Greek symbols indicate the difference from expression levels of Rnc). (c) Analysis of cis -antisense RNA and eno mRNA expression in WT MG1655 cells using RT-PCR. The cDNA was synthesised from the total RNA extracted at each time point using the primers designed to bind cis -antisense RNA and eno mRNA. The PCR products were resolved in an 1.5% agarose gel. Significant differences are indicated with different letters (one-way ANOVA followed by the Student–Newman–Keuls test; Small English letters indicate the difference from expression levels of cis -antisense RNA; Large English letters indicate the difference of expression levels of eno mRNA). (d) Effect of Eno expression levels on the growth of W3110 PBAD eno cells. Cultures of W3110 PBAD eno cells were grown in LB medium containing 0.2% glucose under anaerobic conditions to the early log phase (OD 600 = 0.05) and different concentrations of arabinose (0%, 0.01%, or 0.1%) were added to induce Eno synthesis. As a control, WT W3110 cells were grown in the same way described above and 0.1% arabinose was added. Cultures were further grown (5 h after induction) and were monitored by measuring the OD 600 . Cultures were harvested for western blot analysis of Eno using protein-specific polyclonal antibodies. The grey and blue bars indicate the OD 600 values and the relative expression levels of Eno, respectively. The expression levels of Eno were compared by setting those of W3110 to 1. N.S., not significant. For (b,d) , the S1 protein was used as an internal standard to evaluate the amount of cell extract in each lane. For (c) , the rnpB mRNA was used as an internal standard to evaluate the amount of cell extract in each lane. For (b – d) , the data are presented as means ± s. e. m. of at least three independent experiments.

Techniques Used: Expressing, Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis

Identification of RNase cleavage sites in eno mRNA in vivo . (a) Primer extension analysis of the 5′ UTR of eno mRNA in vivo . Total RNA was isolated from MG1655 strains (WT, Δ rng , Δ rnc , and Δ rnc rng ) and hybridised with the 5′ end 32 P-labelled primer (eno + 52 R). Synthesised cDNA products were separated on a 6% polyacrylamide gel containing 8 M of urea. Sequencing ladders were synthesised with the same primers used for cDNA synthesis and PCR DNA encompassing the eno gene was used as a template. (b) S1 nuclease mapping. Total RNA was hybridised with the 5′ end 32 P-labelled DNA probe. The DNA: RNA complex was treated with 1 U of S1 nuclease and separated in denaturing gel as described above. (c) Predicted eno 5′ UTR secondary structure and RNase cleavage sites. The secondary structure was inferred using the M-fold program. RNase III (1, 2, 3, and 4) cleavage sites identified in (a) and (b) are indicated. The putative Shine–Dalgarno sequence and start codon are indicated as blue and red colours, respectively.
Figure Legend Snippet: Identification of RNase cleavage sites in eno mRNA in vivo . (a) Primer extension analysis of the 5′ UTR of eno mRNA in vivo . Total RNA was isolated from MG1655 strains (WT, Δ rng , Δ rnc , and Δ rnc rng ) and hybridised with the 5′ end 32 P-labelled primer (eno + 52 R). Synthesised cDNA products were separated on a 6% polyacrylamide gel containing 8 M of urea. Sequencing ladders were synthesised with the same primers used for cDNA synthesis and PCR DNA encompassing the eno gene was used as a template. (b) S1 nuclease mapping. Total RNA was hybridised with the 5′ end 32 P-labelled DNA probe. The DNA: RNA complex was treated with 1 U of S1 nuclease and separated in denaturing gel as described above. (c) Predicted eno 5′ UTR secondary structure and RNase cleavage sites. The secondary structure was inferred using the M-fold program. RNase III (1, 2, 3, and 4) cleavage sites identified in (a) and (b) are indicated. The putative Shine–Dalgarno sequence and start codon are indicated as blue and red colours, respectively.

Techniques Used: In Vivo, Isolation, Sequencing, Polymerase Chain Reaction

30) Product Images from "Novel polyubiquitin imaging system, PolyUb-FC, reveals that K33-linked polyubiquitin is recruited by SQSTM1/p62"

Article Title: Novel polyubiquitin imaging system, PolyUb-FC, reveals that K33-linked polyubiquitin is recruited by SQSTM1/p62

Journal: Autophagy

doi: 10.1080/15548627.2017.1407889

SQSTM1/p62 regulates colocalization of K33-linked polyubiquitinated protein to LC3 puncta. (A) Disappearing colocalization of PolyUb(K33)-FC with AsRED-SQSTM1/p62ΔUBA. MEFs were electroporated with PolyUb(K33)-FC vector and AsRED-SQSTM1/p62 (or SQSTM1/p62ΔUBA) plasmid. After 24 h, images were acquired using an FV10i confocal laser microscope. Scale bars: 20 µm. Data are representative of 2 independent experiments. (B) Affinity isolation assay showing ubiquitin chains binding to SQSTM1/p62. Ubiquitin chains bound to negative samples or GST-SQSTM1/p62. Ubiquitin chain input is shown as a control in the left lane of each GST affinity isolation experiment. Input of negative sample and GST-tagged SQSTM1/p62 is shown as a control in the left lanes. Data are representative of 3 independent experiments. (C) Modification of the interaction between K33-linked polyubiquitin chains and SQSTM1/p62 by ZRANB1. HEK293T cells were cotransfected with plasmids encoding MYC-K33Ub, FLAG-SQSTM1/p62, and siRNA against ZRANB1 . After 24 h, cells were lysed and protein extracts were immunoprecipitated (IP) with FLAG antibody and immunoblotted (IB) for the indicated proteins. To confirm the knockdown efficiency of ZRANB1, cells were harvested for RT PCR 24 h after transfection. Data are representative of 2 independent experiments. Error bars indicate standard deviations. (D) Quantification of the number of punctate structures positive for PolyUb(K33)-FC and LC3 per cell. HEK 293T cells were cotransfected with PolyUb(K33)-FC vector and siRNA for SQSTM1/p62 . After 20 h, cells were stained with anti-LC3 antibodies and analyzed by immunofluorescence microscopy. Immunoblots indicate the knockdown efficiency of SQSTM1/p62. In all, 30 cells from each indicated strain were analyzed. Data are representative of 3 independent experiments. *p
Figure Legend Snippet: SQSTM1/p62 regulates colocalization of K33-linked polyubiquitinated protein to LC3 puncta. (A) Disappearing colocalization of PolyUb(K33)-FC with AsRED-SQSTM1/p62ΔUBA. MEFs were electroporated with PolyUb(K33)-FC vector and AsRED-SQSTM1/p62 (or SQSTM1/p62ΔUBA) plasmid. After 24 h, images were acquired using an FV10i confocal laser microscope. Scale bars: 20 µm. Data are representative of 2 independent experiments. (B) Affinity isolation assay showing ubiquitin chains binding to SQSTM1/p62. Ubiquitin chains bound to negative samples or GST-SQSTM1/p62. Ubiquitin chain input is shown as a control in the left lane of each GST affinity isolation experiment. Input of negative sample and GST-tagged SQSTM1/p62 is shown as a control in the left lanes. Data are representative of 3 independent experiments. (C) Modification of the interaction between K33-linked polyubiquitin chains and SQSTM1/p62 by ZRANB1. HEK293T cells were cotransfected with plasmids encoding MYC-K33Ub, FLAG-SQSTM1/p62, and siRNA against ZRANB1 . After 24 h, cells were lysed and protein extracts were immunoprecipitated (IP) with FLAG antibody and immunoblotted (IB) for the indicated proteins. To confirm the knockdown efficiency of ZRANB1, cells were harvested for RT PCR 24 h after transfection. Data are representative of 2 independent experiments. Error bars indicate standard deviations. (D) Quantification of the number of punctate structures positive for PolyUb(K33)-FC and LC3 per cell. HEK 293T cells were cotransfected with PolyUb(K33)-FC vector and siRNA for SQSTM1/p62 . After 20 h, cells were stained with anti-LC3 antibodies and analyzed by immunofluorescence microscopy. Immunoblots indicate the knockdown efficiency of SQSTM1/p62. In all, 30 cells from each indicated strain were analyzed. Data are representative of 3 independent experiments. *p

Techniques Used: Plasmid Preparation, Microscopy, Isolation, Binding Assay, Modification, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Transfection, Staining, Immunofluorescence, Western Blot

SQSTM1/p62 recognizes K33-linked polyubiquitination. (A) Colocalization of PolyUb-FC puncta with aggresomes. HEK 293T cells were transfected with PolyUb-FC vectors. After 20 h, aggresomes in the cells were stained. Images were acquired using an FV10i confocal laser microscope every 3 min. Data are representative of 4 independent experiments. Scale bars: 10 μm. Data are representative of 2 independent experiments. (B) Colocalization of Poly-Ub(K33)-FC with endogenous SQSTM1/p62. HEK 293T cells were cotransfected with PolyUb-FC vectors. After 20 h, cells were stained with anti-SQSTM1/p62 antibodies. Images were acquired using an FV10i confocal laser microscope. Scale bars: 10 µm. Data are representative of 2 independent experiments. (C) Recruitment of PolyUb(K33)-FC to FLAG-SQSTM1/p62. HEK 293T cells were cotransfected with PolyUb-FC vectors and a plasmid encoding FLAG-SQSTM1/p62. After 20 h, cells were lysed and protein extracts were immunoprecipitated (IP) with FLAG antibody and immunoblotted (IB) for the indicated proteins. Data are representative of 3 independent experiments. (D) Interaction of MYC-K33Ub and FLAG-SQSTM1/p62. HEK 293T cells were cotransfected with plasmids encoding MYC-K33Ub and FLAG-SQSTM1/p62. Twenty h later, protein extracts were immunoprecipitated (IP) with FLAG antibody and immunoblotted for the indicated proteins. Data are representative of 2 independent experiments. (E) Recruitment of MYC-K33Ub to endogenous SQSTM1/p62. HEK 293T cells were transfected with a plasmid encoding MYC-K33Ub. After 20 h, cells were lysed and protein extracts were immunoprecipitated with MYC antibody and immunoblotted (IB) for the indicated proteins. Data are representative of 3 independent experiments.
Figure Legend Snippet: SQSTM1/p62 recognizes K33-linked polyubiquitination. (A) Colocalization of PolyUb-FC puncta with aggresomes. HEK 293T cells were transfected with PolyUb-FC vectors. After 20 h, aggresomes in the cells were stained. Images were acquired using an FV10i confocal laser microscope every 3 min. Data are representative of 4 independent experiments. Scale bars: 10 μm. Data are representative of 2 independent experiments. (B) Colocalization of Poly-Ub(K33)-FC with endogenous SQSTM1/p62. HEK 293T cells were cotransfected with PolyUb-FC vectors. After 20 h, cells were stained with anti-SQSTM1/p62 antibodies. Images were acquired using an FV10i confocal laser microscope. Scale bars: 10 µm. Data are representative of 2 independent experiments. (C) Recruitment of PolyUb(K33)-FC to FLAG-SQSTM1/p62. HEK 293T cells were cotransfected with PolyUb-FC vectors and a plasmid encoding FLAG-SQSTM1/p62. After 20 h, cells were lysed and protein extracts were immunoprecipitated (IP) with FLAG antibody and immunoblotted (IB) for the indicated proteins. Data are representative of 3 independent experiments. (D) Interaction of MYC-K33Ub and FLAG-SQSTM1/p62. HEK 293T cells were cotransfected with plasmids encoding MYC-K33Ub and FLAG-SQSTM1/p62. Twenty h later, protein extracts were immunoprecipitated (IP) with FLAG antibody and immunoblotted for the indicated proteins. Data are representative of 2 independent experiments. (E) Recruitment of MYC-K33Ub to endogenous SQSTM1/p62. HEK 293T cells were transfected with a plasmid encoding MYC-K33Ub. After 20 h, cells were lysed and protein extracts were immunoprecipitated with MYC antibody and immunoblotted (IB) for the indicated proteins. Data are representative of 3 independent experiments.

Techniques Used: Transfection, Staining, Microscopy, Plasmid Preparation, Immunoprecipitation

Generation of iPolyUb-FC plasmids and validation of their function. (A) Generation of iPolyUb-FC vector. Schematic represents iPolyUb-FC vector plasmids encoding mKG(C)-Ub-IRES-mKG(N)-Ub. mKG(N)-Ub-IRES-mKG(C)-Ub coding sequences were cloned into the pTet-One vector from PolyUb(FC). HEK 293T cells were transfected with iPolyUb(K0)-FC and iPolyUb(WT)-FC. After 24 h, images were acquired using an FV10i confocal laser microscope. Scale bars: 10 µm. HEK 293T cells were transfected with PolyUb(WT)-FC and iPolyUb-FC vectors. After 24 h, cells were lysed and immunoblotted. The Dox concentrations used are indicated. Induction was for 24 h. Data are representative of 2 independent experiments. (B) Colocalization of iPoly-Ub-FC vectors with endogenous SQSTM1/p62. HEK 293T cells were cotransfected with iPolyUb-FC vectors. After 24 h, cells were stained with anti-SQSTM1/p62 antibodies. The Dox concentrations used are indicated. Induction was for 24 h. Images were acquired using an FV10i confocal laser microscope. Scale bars: 10 µm. Data are representative of 2 independent experiments. (C) Quantification of the number of punctate structures positive for iPolyUb(K33)-FC and LC3 per cell. HEK 293T cells were cotransfected with iPolyUb(K33)-FC vector and siRNA for SQSTM1/p62 . After 24 h, cells were stained with anti-LC3 antibodies and analyzed by immunofluorescence microscopy. The Dox concentration used was 100 ng/ml. Induction was for 24 h. In all, 30 cells from each indicated strain were analyzed. Data are representative of 2 independent experiments. *p
Figure Legend Snippet: Generation of iPolyUb-FC plasmids and validation of their function. (A) Generation of iPolyUb-FC vector. Schematic represents iPolyUb-FC vector plasmids encoding mKG(C)-Ub-IRES-mKG(N)-Ub. mKG(N)-Ub-IRES-mKG(C)-Ub coding sequences were cloned into the pTet-One vector from PolyUb(FC). HEK 293T cells were transfected with iPolyUb(K0)-FC and iPolyUb(WT)-FC. After 24 h, images were acquired using an FV10i confocal laser microscope. Scale bars: 10 µm. HEK 293T cells were transfected with PolyUb(WT)-FC and iPolyUb-FC vectors. After 24 h, cells were lysed and immunoblotted. The Dox concentrations used are indicated. Induction was for 24 h. Data are representative of 2 independent experiments. (B) Colocalization of iPoly-Ub-FC vectors with endogenous SQSTM1/p62. HEK 293T cells were cotransfected with iPolyUb-FC vectors. After 24 h, cells were stained with anti-SQSTM1/p62 antibodies. The Dox concentrations used are indicated. Induction was for 24 h. Images were acquired using an FV10i confocal laser microscope. Scale bars: 10 µm. Data are representative of 2 independent experiments. (C) Quantification of the number of punctate structures positive for iPolyUb(K33)-FC and LC3 per cell. HEK 293T cells were cotransfected with iPolyUb(K33)-FC vector and siRNA for SQSTM1/p62 . After 24 h, cells were stained with anti-LC3 antibodies and analyzed by immunofluorescence microscopy. The Dox concentration used was 100 ng/ml. Induction was for 24 h. In all, 30 cells from each indicated strain were analyzed. Data are representative of 2 independent experiments. *p

Techniques Used: Plasmid Preparation, Clone Assay, Transfection, Microscopy, Staining, Immunofluorescence, Concentration Assay

31) Product Images from "Decreased endothelin receptor B expression in large primary uveal melanomas is associated with early clinical metastasis and short survival"

Article Title: Decreased endothelin receptor B expression in large primary uveal melanomas is associated with early clinical metastasis and short survival

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6600620

Comparative multiplex RT–PCR of EDNRB and the control gene KIAA0228 in SCLCs. N: cDNA from matched normal tissue. T: Tumour cDNA.
Figure Legend Snippet: Comparative multiplex RT–PCR of EDNRB and the control gene KIAA0228 in SCLCs. N: cDNA from matched normal tissue. T: Tumour cDNA.

Techniques Used: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction

32) Product Images from "A gonococcal homologue of meningococcal ?-glutamyl transpeptidase gene is a new type of bacterial pseudogene that is transcriptionally active but phenotypically silent"

Article Title: A gonococcal homologue of meningococcal ?-glutamyl transpeptidase gene is a new type of bacterial pseudogene that is transcriptionally active but phenotypically silent

Journal: BMC Microbiology

doi: 10.1186/1471-2180-5-56

Transcriptional expression of the ggh genes. A. Dot blot analysis using the meningococcal ggt gene as a probe. One to 16 micrograms of RNAs isolated from H44/76, HT1089 (H44/76 Δggt::spc ) [39], ATCC49226, NIID54, NIID103 and NIID106 were subjected to this analysis. B. RT-PCR to detect the transcripts of the gonococcal ggh genes. The schematic figure in the box depicts the position of the primers used in this experiment (see Table 1). RT-PCR was performed without reverse transcriptase (RTase) (lanes 1 to 5) or with RTase (lanes 6 to 10). Lanes 1 and 6, H44/76 ( N. meningitidis ); lanes 2 and 7, ATCC49226 ( N. gonorrhoeae ); lanes 3 and 8, NIID54 ( N. gonorrhoeae ); lanes 4 and 9, NIID103 ( N. gonorrhoeae ); lanes 5 and 10, NIID106 ( N. gonorrhoeae ). The marker in the left-most lane is φ X174 DNA digested with Hae III. Primer sets used for RT-PCR are shown on the left side, and the corresponding PCR products are indicated by arrows on the right side. C. Primer extension analysis to detect the transcriptional start point of the ggt and ggh genes. Total RNA extracted from H44/76 ( N. meningitidis ), ATCC49226 and NIID106 ( N. gonorrhoeae ) was used for the primer extension with AMV reverse transcriptase XL and biotin-labeled oligonucleotide ggt-ext-2. The arrow on the right side indicates the transcriptional start site. D. Alignment of the nucleotide sequences of the upstream regions of the ggt and ggh genes. The sequence data have been deposited in the DDBJ/EMBL/GenBank Databases under the following Accession Numbers: N. meningitidis strains H44/76 [DDBJ: AB193252 ], H114/90 [DDBJ: AB193253 ], N. gonorrhoeae strains ATCC49226 [DDBJ: AB193254 ], NIID54 [DDBJ: AB193255 ], NIID103 [DDBJ: AB193296 ], NIID106 [DDBJ: AB193256 ]. An identical nucleotide is represented as *. The transcriptional start site is shown in bold as +1. The putative -35, -10 elements and Shine-Dalgarno sequence (SD) are depicted in the box, and the ideal -35 and -10 nucleotide sequences are shown above the boxes. The previously predicted start codon (ATG) [25] and newly predicted start codon (GTG) of the meningococcal {\it ggt} gene are underlined. The amino acid sequence deduced from the putative start codon GTG (shown in bold) in the meningococcal {\it ggt} gene is also shown under the corresponding nucleotide sequences.
Figure Legend Snippet: Transcriptional expression of the ggh genes. A. Dot blot analysis using the meningococcal ggt gene as a probe. One to 16 micrograms of RNAs isolated from H44/76, HT1089 (H44/76 Δggt::spc ) [39], ATCC49226, NIID54, NIID103 and NIID106 were subjected to this analysis. B. RT-PCR to detect the transcripts of the gonococcal ggh genes. The schematic figure in the box depicts the position of the primers used in this experiment (see Table 1). RT-PCR was performed without reverse transcriptase (RTase) (lanes 1 to 5) or with RTase (lanes 6 to 10). Lanes 1 and 6, H44/76 ( N. meningitidis ); lanes 2 and 7, ATCC49226 ( N. gonorrhoeae ); lanes 3 and 8, NIID54 ( N. gonorrhoeae ); lanes 4 and 9, NIID103 ( N. gonorrhoeae ); lanes 5 and 10, NIID106 ( N. gonorrhoeae ). The marker in the left-most lane is φ X174 DNA digested with Hae III. Primer sets used for RT-PCR are shown on the left side, and the corresponding PCR products are indicated by arrows on the right side. C. Primer extension analysis to detect the transcriptional start point of the ggt and ggh genes. Total RNA extracted from H44/76 ( N. meningitidis ), ATCC49226 and NIID106 ( N. gonorrhoeae ) was used for the primer extension with AMV reverse transcriptase XL and biotin-labeled oligonucleotide ggt-ext-2. The arrow on the right side indicates the transcriptional start site. D. Alignment of the nucleotide sequences of the upstream regions of the ggt and ggh genes. The sequence data have been deposited in the DDBJ/EMBL/GenBank Databases under the following Accession Numbers: N. meningitidis strains H44/76 [DDBJ: AB193252 ], H114/90 [DDBJ: AB193253 ], N. gonorrhoeae strains ATCC49226 [DDBJ: AB193254 ], NIID54 [DDBJ: AB193255 ], NIID103 [DDBJ: AB193296 ], NIID106 [DDBJ: AB193256 ]. An identical nucleotide is represented as *. The transcriptional start site is shown in bold as +1. The putative -35, -10 elements and Shine-Dalgarno sequence (SD) are depicted in the box, and the ideal -35 and -10 nucleotide sequences are shown above the boxes. The previously predicted start codon (ATG) [25] and newly predicted start codon (GTG) of the meningococcal {\it ggt} gene are underlined. The amino acid sequence deduced from the putative start codon GTG (shown in bold) in the meningococcal {\it ggt} gene is also shown under the corresponding nucleotide sequences.

Techniques Used: Expressing, Dot Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Marker, Polymerase Chain Reaction, Labeling, Sequencing

33) Product Images from "Curcumin analog, GO‐Y078, overcomes resistance to tumor angiogenesis inhibitors, et al. Curcumin analog, GO‐Y078, overcomes resistance to tumor angiogenesis inhibitors"

Article Title: Curcumin analog, GO‐Y078, overcomes resistance to tumor angiogenesis inhibitors, et al. Curcumin analog, GO‐Y078, overcomes resistance to tumor angiogenesis inhibitors

Journal: Cancer Science

doi: 10.1111/cas.13741

Knockdown effect of fibronectin 1 ( FN1 ) on the growth of HUVECKi2. A, Efficacies of Si‐oligos to FN1 mRNA in the attached HUVECKi2. B, Effects on the growth of the attached HUVECKi2 treated with FN 1 ‐Si‐oligo (open circle), control‐Si‐oligo (closed rectangle), and without Si‐oligo (closed circle). C, Efficacies of Si‐oligos to FN 1 mRNA in the suspended HUVECKi2. D, Effects of the growth of the suspended HUVECKi2 treated with the FN 1 ‐Si‐oligo (open circle), control‐Si‐oligo (closed rectangle), and without Si‐oligo (closed circle)
Figure Legend Snippet: Knockdown effect of fibronectin 1 ( FN1 ) on the growth of HUVECKi2. A, Efficacies of Si‐oligos to FN1 mRNA in the attached HUVECKi2. B, Effects on the growth of the attached HUVECKi2 treated with FN 1 ‐Si‐oligo (open circle), control‐Si‐oligo (closed rectangle), and without Si‐oligo (closed circle). C, Efficacies of Si‐oligos to FN 1 mRNA in the suspended HUVECKi2. D, Effects of the growth of the suspended HUVECKi2 treated with the FN 1 ‐Si‐oligo (open circle), control‐Si‐oligo (closed rectangle), and without Si‐oligo (closed circle)

Techniques Used:

34) Product Images from "Chicken GHR natural antisense transcript regulates GHR mRNA in LMH cells"

Article Title: Chicken GHR natural antisense transcript regulates GHR mRNA in LMH cells

Journal: Oncotarget

doi: 10.18632/oncotarget.12437

GHR-AS and GHR-S can form double strand RNAs at the last exon of GHR gene ( A ) DNA contamination detection, using β-actin gene, demonstrated the RNAs were DNA-free. 1: positive control using DNA as template for PCR; 2,3: cDNA obtained from RNA of LMH cells; 4,5: cDNA obtained from RNA of chicken liver. ( B ) RT-PCR with gene specific primers, located in exon6 and 7 of GHR gene, suggested no production detected. 1: positive control using cDNA as template for PCR; 2,3: cDNA obtained from LMH cells RNA treated with RNase A; 4,5: cDNA obtained from chicken liver RNA treated with RNase A. ( C ) The GHR-S and GHR-AS RNA hybrids were detected by RT-PCR with gene specific primers, located in exon8 of GHR gene. The templates used were the same to (B).
Figure Legend Snippet: GHR-AS and GHR-S can form double strand RNAs at the last exon of GHR gene ( A ) DNA contamination detection, using β-actin gene, demonstrated the RNAs were DNA-free. 1: positive control using DNA as template for PCR; 2,3: cDNA obtained from RNA of LMH cells; 4,5: cDNA obtained from RNA of chicken liver. ( B ) RT-PCR with gene specific primers, located in exon6 and 7 of GHR gene, suggested no production detected. 1: positive control using cDNA as template for PCR; 2,3: cDNA obtained from LMH cells RNA treated with RNase A; 4,5: cDNA obtained from chicken liver RNA treated with RNase A. ( C ) The GHR-S and GHR-AS RNA hybrids were detected by RT-PCR with gene specific primers, located in exon8 of GHR gene. The templates used were the same to (B).

Techniques Used: Positive Control, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

35) Product Images from "Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array"

Article Title: Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array

Journal: Nucleic Acids Research

doi:

Phenotypes, experimental procedures for and results of PCR-select cDNA subtraction. ( A ) Seeds of wild-type upland cotton Xuzhou 142 (left) and fl (fuzzless-lintless) mutant (right). ( B ) Strategy for isolating and identifying cotton fiber development-related genes via PCR-select cDNA subtraction and differential screening. ( C ) Products of PCR-select cDNA subtraction. M, Φ174/ Hae III digested molecular marker; UD, unsubtracted driver cDNA; UT, unsubtracted tester cDNA; S1, PCR products obtained after one round of subtraction; S2, PCR products obtained after two rounds of subtraction.
Figure Legend Snippet: Phenotypes, experimental procedures for and results of PCR-select cDNA subtraction. ( A ) Seeds of wild-type upland cotton Xuzhou 142 (left) and fl (fuzzless-lintless) mutant (right). ( B ) Strategy for isolating and identifying cotton fiber development-related genes via PCR-select cDNA subtraction and differential screening. ( C ) Products of PCR-select cDNA subtraction. M, Φ174/ Hae III digested molecular marker; UD, unsubtracted driver cDNA; UT, unsubtracted tester cDNA; S1, PCR products obtained after one round of subtraction; S2, PCR products obtained after two rounds of subtraction.

Techniques Used: Polymerase Chain Reaction, Mutagenesis, Marker

Differential screening of the 280 cDNAs by macroarray, using fiber cDNA (left) and fl ovule cDNA (right) as probes. A cotton ubiquitin (P2D01) gene was used as the positive control. Negative controls include PCR primers (P1D09), ddH 2 O (P1B07) and vector DNA (P1D10).
Figure Legend Snippet: Differential screening of the 280 cDNAs by macroarray, using fiber cDNA (left) and fl ovule cDNA (right) as probes. A cotton ubiquitin (P2D01) gene was used as the positive control. Negative controls include PCR primers (P1D09), ddH 2 O (P1B07) and vector DNA (P1D10).

Techniques Used: Positive Control, Polymerase Chain Reaction, Plasmid Preparation

RT–PCR analysis of cotton fiber cDNAs. Total RNA (5 µg) isolated from fl mutant ovules and from fiber cells at different developmental stages was used to synthesize first-strand cDNA for RT–PCR using gene-specific primers. A cotton ubiquitin cDNA was used to normalize the amount of templates added in PCR reactions. M, fl mutant ovule; UF, upland cotton fiber. Days post anthesis (d.p.a., days 0–20) are shown at the top. Capitalized gene names indicate previously reported fiber genes that were not recovered from our subtractive library. These genes did not show significant differences in expression between 10 d.p.a. fiber and fl mutant ovules. KCBP, kinesin-like calmodulin binding protein; VLCFA, very-long chain fatty acid; CAP, adenyl cyclase associated protein; RGP, reversibly glycosylated polypeptide.
Figure Legend Snippet: RT–PCR analysis of cotton fiber cDNAs. Total RNA (5 µg) isolated from fl mutant ovules and from fiber cells at different developmental stages was used to synthesize first-strand cDNA for RT–PCR using gene-specific primers. A cotton ubiquitin cDNA was used to normalize the amount of templates added in PCR reactions. M, fl mutant ovule; UF, upland cotton fiber. Days post anthesis (d.p.a., days 0–20) are shown at the top. Capitalized gene names indicate previously reported fiber genes that were not recovered from our subtractive library. These genes did not show significant differences in expression between 10 d.p.a. fiber and fl mutant ovules. KCBP, kinesin-like calmodulin binding protein; VLCFA, very-long chain fatty acid; CAP, adenyl cyclase associated protein; RGP, reversibly glycosylated polypeptide.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Mutagenesis, Polymerase Chain Reaction, Expressing, Binding Assay

36) Product Images from "cDNA Arrays and Immunohistochemistry Identification of CD10/CALLA Expression in Hepatocellular Carcinoma"

Article Title: cDNA Arrays and Immunohistochemistry Identification of CD10/CALLA Expression in Hepatocellular Carcinoma

Journal: The American Journal of Pathology

doi:

A: Differential mRNA expression profiles between HCC and adjacent nonneoplastic liver tissue (NT) as monitored using an Atlas Human cDNA 1.2II microarray. A representative section of the array is shown in which differential mRNA expression of CD10 is indicated by arrows . B: Up-regulation of CD10 mRNA expression as confirmed by RT-PCR. Semiquantitative RT-PCR was performed using specific primers for CD10 and total cellular RNA isolated from tissue surrounding the HCC (NT) and the HCC from two patients (P1 and P2). Co-amplification of human GAPDH was used as an internal control. C: Increased CD10 protein expression in HCC. Total cellular extracts from tissue surrounding the HCC (NT) and HCC were prepared from patient P1, and separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis and CD10 was identified by immunoblotting with anti-CD10 antibody. A single species at ∼100 kd is seen, consistent with the reported molecular weight of CD10.
Figure Legend Snippet: A: Differential mRNA expression profiles between HCC and adjacent nonneoplastic liver tissue (NT) as monitored using an Atlas Human cDNA 1.2II microarray. A representative section of the array is shown in which differential mRNA expression of CD10 is indicated by arrows . B: Up-regulation of CD10 mRNA expression as confirmed by RT-PCR. Semiquantitative RT-PCR was performed using specific primers for CD10 and total cellular RNA isolated from tissue surrounding the HCC (NT) and the HCC from two patients (P1 and P2). Co-amplification of human GAPDH was used as an internal control. C: Increased CD10 protein expression in HCC. Total cellular extracts from tissue surrounding the HCC (NT) and HCC were prepared from patient P1, and separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis and CD10 was identified by immunoblotting with anti-CD10 antibody. A single species at ∼100 kd is seen, consistent with the reported molecular weight of CD10.

Techniques Used: Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Polyacrylamide Gel Electrophoresis, Molecular Weight

37) Product Images from "Disturbed Wnt Signalling due to a Mutation in CCDC88C Causes an Autosomal Recessive Non-Syndromic Hydrocephalus with Medial Diverticulum"

Article Title: Disturbed Wnt Signalling due to a Mutation in CCDC88C Causes an Autosomal Recessive Non-Syndromic Hydrocephalus with Medial Diverticulum

Journal: Molecular Syndromology

doi: 10.1159/000319859

CCDC88C expression studies by quantitative RT-PCR in cDNA from various human fetal and adult tissues showed highest relative expression in fetal brain and highest relative expression in adult pancreas ( A ). B Upper part represents a scheme of the various
Figure Legend Snippet: CCDC88C expression studies by quantitative RT-PCR in cDNA from various human fetal and adult tissues showed highest relative expression in fetal brain and highest relative expression in adult pancreas ( A ). B Upper part represents a scheme of the various

Techniques Used: Expressing, Quantitative RT-PCR

38) Product Images from "P2Y-like receptor, GPR105 (P2Y14), identifies and mediates chemotaxis of bone-marrowhematopoietic stem cells"

Article Title: P2Y-like receptor, GPR105 (P2Y14), identifies and mediates chemotaxis of bone-marrowhematopoietic stem cells

Journal: Genes & Development

doi: 10.1101/gad.1071503

GPR105 is restricted in tissue expression and within hematopoietic cells is limited to primitive cells. ( A ) A human tissue blot with indicated mRNAs was probed with radiolabeled GPR105 cDNA and hybridization determined by autoradiography. Size markers are indicated in kilobases. ( B ) Adult human cell mRNA from cells bearing the indicated phenotype was assessed by poly(A)-primed RT–PCR and the resulting cDNA was probed with either GPR105 or GAPDH sequences. Cells labeled G0 CD34 + CD38 – ) and represent
Figure Legend Snippet: GPR105 is restricted in tissue expression and within hematopoietic cells is limited to primitive cells. ( A ) A human tissue blot with indicated mRNAs was probed with radiolabeled GPR105 cDNA and hybridization determined by autoradiography. Size markers are indicated in kilobases. ( B ) Adult human cell mRNA from cells bearing the indicated phenotype was assessed by poly(A)-primed RT–PCR and the resulting cDNA was probed with either GPR105 or GAPDH sequences. Cells labeled G0 CD34 + CD38 – ) and represent

Techniques Used: Expressing, Hybridization, Autoradiography, Reverse Transcription Polymerase Chain Reaction, Labeling

Anti-GPR105 recognizes GPR105 and identifies a subset of CD34 + CD38 – fetal bone marrow cells. ( A ) HA-epitope-tagged GPR105 cDNA was in vitro transcribed and translated. ( Left ) In vitro translated protein was immunoprecipitated with anti-GPR105 antibody. Note that the increase of the size in NC-tagged HA is caused by an additional tagging. ( Right ) Similar results were obtained using anti-HA tag monoclonal antibody for immunoprecipitation. PcDNA without GPR105 was used as a negative control. N-HA, N terminus-tagged; C-HA, C terminus-tagged; NC-HA, N and C terminus-tagged. The position of a molecular weight marker (size in kilodaltons) is indicated to the left . ( B ) Flow cytometry of cells transduced with either MSCV control (blue line) or MSCV-GPR105 (red line) vectors and stained with affinity-purified anti-GPR105. ( C ) CD34 + CD38 – GPR105 + human fetal bone marrow cells express GPR105 mRNA as demonstrated by semiquantitative RT–PCR. Cells isolated by cell sorting were assessed using 3 × 10 3 cells of each type and analyzed for GPR105 or actin mRNA; control refers to a no-cDNA template. ( D ) CD34 + CD38 – fetal bone marrow cells stained with anti-GPR105-FITC or control antiserum from a single experiment ( top panels) or composite data from four independent experiments (table).
Figure Legend Snippet: Anti-GPR105 recognizes GPR105 and identifies a subset of CD34 + CD38 – fetal bone marrow cells. ( A ) HA-epitope-tagged GPR105 cDNA was in vitro transcribed and translated. ( Left ) In vitro translated protein was immunoprecipitated with anti-GPR105 antibody. Note that the increase of the size in NC-tagged HA is caused by an additional tagging. ( Right ) Similar results were obtained using anti-HA tag monoclonal antibody for immunoprecipitation. PcDNA without GPR105 was used as a negative control. N-HA, N terminus-tagged; C-HA, C terminus-tagged; NC-HA, N and C terminus-tagged. The position of a molecular weight marker (size in kilodaltons) is indicated to the left . ( B ) Flow cytometry of cells transduced with either MSCV control (blue line) or MSCV-GPR105 (red line) vectors and stained with affinity-purified anti-GPR105. ( C ) CD34 + CD38 – GPR105 + human fetal bone marrow cells express GPR105 mRNA as demonstrated by semiquantitative RT–PCR. Cells isolated by cell sorting were assessed using 3 × 10 3 cells of each type and analyzed for GPR105 or actin mRNA; control refers to a no-cDNA template. ( D ) CD34 + CD38 – fetal bone marrow cells stained with anti-GPR105-FITC or control antiserum from a single experiment ( top panels) or composite data from four independent experiments (table).

Techniques Used: In Vitro, Immunoprecipitation, Negative Control, Molecular Weight, Marker, Flow Cytometry, Cytometry, Transduction, Staining, Affinity Purification, Reverse Transcription Polymerase Chain Reaction, Isolation, FACS

39) Product Images from "PIM-1 contributes to the malignancy of pancreatic cancer and displays diagnostic and prognostic value"

Article Title: PIM-1 contributes to the malignancy of pancreatic cancer and displays diagnostic and prognostic value

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-016-0406-z

PIM-1 downregulation decreased the expression levels of cancer stem cell markers in pancreatic cancer. qRT-PCR was used to detect the mRNA expression levels of the indicated cancer stem cell markers. GAPDH was used as an internal control. The data are displayed as the mean ± SD (*, P
Figure Legend Snippet: PIM-1 downregulation decreased the expression levels of cancer stem cell markers in pancreatic cancer. qRT-PCR was used to detect the mRNA expression levels of the indicated cancer stem cell markers. GAPDH was used as an internal control. The data are displayed as the mean ± SD (*, P

Techniques Used: Expressing, Quantitative RT-PCR

40) Product Images from "Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate"

Article Title: Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200202052

Interaction between MISS and MAPK. (A) Two-hybrid interaction between MISS and MAPK. MISS interacts with ERK2WT and ERK2KD but not with a negative control. The yeast transformed with LexA-ERK2WT, LexA-ERK2KD ( Waskiewicz et al., 1997 ), LexA-53 (positive control; CLONTECH Laboratories, Inc.), LexA-Su(Fu), or LexA alone were mated with yeast transformed respectively with B42-cDNA, B42-AD-T (positive control; CLONTECH Laboratories, Inc.), or empty B42. The diploids obtained were assayed for transactivation of both the β-galactosidase and the LEU2 reporter genes on glucose (Glu)- or galactose (Gal/Raf)-containing mediums. The B42 fusion proteins are under the control of the galactose promoter. The B42-cDNA clearly interacts both with fusions of ERK2WT and ERK2KD at levels identical to the positive control, whereas it does not interact with a negative control (Su[Fu]). (B) MISS coimmunoprecipitates with endogenous p42 mpk from Xenopus oocyte extracts. Oocyte extracts expressing Myc–Wnt11 ( Xenopus Wint11; negative control) or Myc–MISS RNA (lanes 1 and 2). Anti-p42 mpk1 immunoprecipitates (lanes 3 and 4) prepared from the oocyte lysates were analyzed by immunoblotting with anti-Myc antibody. This experiment was performed three times. (C) Expression of MISS RNA in immature oocytes and two cell stage embryos. For RT-PCR analysis, total RNA was isolated from mouse ovaries (lane 2), immature oocytes (lanes 3 and 4), and two cell stage embryos (lanes 5 and 6) and treated with or without reverse transcriptase (RT, + or −). Lane 1 corresponds to a PCR control (water). (D) MISS accumulates during meiotic maturation. 2,000 immature (lane 1) or mature (lane 2) oocytes were collected and immunoblotted using an affinity-purified anti-MISS antibody.
Figure Legend Snippet: Interaction between MISS and MAPK. (A) Two-hybrid interaction between MISS and MAPK. MISS interacts with ERK2WT and ERK2KD but not with a negative control. The yeast transformed with LexA-ERK2WT, LexA-ERK2KD ( Waskiewicz et al., 1997 ), LexA-53 (positive control; CLONTECH Laboratories, Inc.), LexA-Su(Fu), or LexA alone were mated with yeast transformed respectively with B42-cDNA, B42-AD-T (positive control; CLONTECH Laboratories, Inc.), or empty B42. The diploids obtained were assayed for transactivation of both the β-galactosidase and the LEU2 reporter genes on glucose (Glu)- or galactose (Gal/Raf)-containing mediums. The B42 fusion proteins are under the control of the galactose promoter. The B42-cDNA clearly interacts both with fusions of ERK2WT and ERK2KD at levels identical to the positive control, whereas it does not interact with a negative control (Su[Fu]). (B) MISS coimmunoprecipitates with endogenous p42 mpk from Xenopus oocyte extracts. Oocyte extracts expressing Myc–Wnt11 ( Xenopus Wint11; negative control) or Myc–MISS RNA (lanes 1 and 2). Anti-p42 mpk1 immunoprecipitates (lanes 3 and 4) prepared from the oocyte lysates were analyzed by immunoblotting with anti-Myc antibody. This experiment was performed three times. (C) Expression of MISS RNA in immature oocytes and two cell stage embryos. For RT-PCR analysis, total RNA was isolated from mouse ovaries (lane 2), immature oocytes (lanes 3 and 4), and two cell stage embryos (lanes 5 and 6) and treated with or without reverse transcriptase (RT, + or −). Lane 1 corresponds to a PCR control (water). (D) MISS accumulates during meiotic maturation. 2,000 immature (lane 1) or mature (lane 2) oocytes were collected and immunoblotted using an affinity-purified anti-MISS antibody.

Techniques Used: Negative Control, Transformation Assay, Positive Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Affinity Purification

Related Articles

Selection:

Article Title: Assessment of an Organ-Specific de Novo Transcriptome of the Nematode Trap-Crop, Solanum sisymbriifolium
Article Snippet: .. The Iso-Seq libraries for four organs, root, stem, leaf and bud, were prepared for Isoform Sequencing (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol as described by Pacific Biosciences ( https://goo.gl/ij71Hh ) with the following modifications. .. For cDNA conversion, 3 µg of total RNA was put into each Clonetech SMARTer reaction.

Article Title: Long-read assays shed new light on the transcriptome complexity of a viral pathogen
Article Snippet: .. cDNA synthesisCopy DNAs were generated from polyA(+) RNA fractions using SMARTer PCR cDNA Synthesis Kits (Clontech) according to ‘PacBio Isoform Sequencing (Iso-Seq) using Clontech SMARTer PCR cDNA Synthesis Kit and No Size Selection’ protocols. .. Samples from different infection time points (1-, 4-, 8-, and 12-h p.i.) were mixed for RSII library preparation, whereas 1-, 2-, 3-, 4-, 6-, and 8-h p.i. samples were mixed for Sequel sequencing.

Agarose Gel Electrophoresis:

Article Title: Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica *
Article Snippet: .. High yields of full-length cDNAs were generated from 1 μg total RNA of normal and fibrosis HSCs using SMART PCR cDNA synthesis kit (Clontech, USA). cDNAs (0.2 μg) were electrophosed on a 1.0% agarose gel, transferred onto Hybond N+ Membrane (Roch, Germany) and hybridized with DIG-labelled clone probes. .. These probes were generated by PCR amplification of the relevant inserts, adaptor-cutting and column-purification.

Sequencing:

Article Title: Assessment of an Organ-Specific de Novo Transcriptome of the Nematode Trap-Crop, Solanum sisymbriifolium
Article Snippet: .. The Iso-Seq libraries for four organs, root, stem, leaf and bud, were prepared for Isoform Sequencing (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol as described by Pacific Biosciences ( https://goo.gl/ij71Hh ) with the following modifications. .. For cDNA conversion, 3 µg of total RNA was put into each Clonetech SMARTer reaction.

Article Title: Long-read assays shed new light on the transcriptome complexity of a viral pathogen
Article Snippet: .. cDNA synthesisCopy DNAs were generated from polyA(+) RNA fractions using SMARTer PCR cDNA Synthesis Kits (Clontech) according to ‘PacBio Isoform Sequencing (Iso-Seq) using Clontech SMARTer PCR cDNA Synthesis Kit and No Size Selection’ protocols. .. Samples from different infection time points (1-, 4-, 8-, and 12-h p.i.) were mixed for RSII library preparation, whereas 1-, 2-, 3-, 4-, 6-, and 8-h p.i. samples were mixed for Sequel sequencing.

cDNA Library Assay:

Article Title: PR-10, defensin and cold dehydrin genes are among those over expressed in Oxytropis (Fabaceae) species adapted to the arctic
Article Snippet: .. Suppressive subtraction cDNA library construction Extracted RNA from two plantlets of a species were pooled prior to complimentary DNA (cDNA) synthesis, and 1 mg of this pool was used as template for cDNA synthesis using the SuperSmart PCR cDNA Synthesis kit (Clontech, Mountain View, CA). .. Genes specifically expressed in either the arctic or in the temperate species were isolated by applying the suppression subtractive hybridization strategy (Diatchenko et al. ) using the PCR-select cDNA subtraction kit (Clontech, Mountain View, CA).

Generated:

Article Title: Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica *
Article Snippet: .. High yields of full-length cDNAs were generated from 1 μg total RNA of normal and fibrosis HSCs using SMART PCR cDNA synthesis kit (Clontech, USA). cDNAs (0.2 μg) were electrophosed on a 1.0% agarose gel, transferred onto Hybond N+ Membrane (Roch, Germany) and hybridized with DIG-labelled clone probes. .. These probes were generated by PCR amplification of the relevant inserts, adaptor-cutting and column-purification.

Article Title: Identification of differentially expressed genes in normal mucosa, adenoma and adenocarcinoma of colon by SSH
Article Snippet: .. High yields of full-length cDNAs were generated from 1 μg total RNA of normal mucosa, adenoma and adenocarcinoma using SMART PCR cDNA Synthesis kit (Clontech, USA). .. 0.2 μg cDNAs were electrop hosed on a 1.0% agarose gel, transferred onto Hybond N+ Membrane (Boeheringer Mannheim, Germany) and hybridized with DIG-labeled clone probes.

Article Title: A novel method to isolate the common fraction of two DNA samples: hybrid specific amplification (HSA)
Article Snippet: .. Double-stranded cDNA was generated with the SMART PCR cDNA Synthesis Kit (Clontech). .. Aliquots of 2 µg of Qiagen-prepared BAC DNA and 1 µg of SMART cDNA PCR product were digested by Rsa I (15 U) for 90 min at 37°C.

Article Title: Long-read assays shed new light on the transcriptome complexity of a viral pathogen
Article Snippet: .. cDNA synthesisCopy DNAs were generated from polyA(+) RNA fractions using SMARTer PCR cDNA Synthesis Kits (Clontech) according to ‘PacBio Isoform Sequencing (Iso-Seq) using Clontech SMARTer PCR cDNA Synthesis Kit and No Size Selection’ protocols. .. Samples from different infection time points (1-, 4-, 8-, and 12-h p.i.) were mixed for RSII library preparation, whereas 1-, 2-, 3-, 4-, 6-, and 8-h p.i. samples were mixed for Sequel sequencing.

Polymerase Chain Reaction:

Article Title: Assessment of an Organ-Specific de Novo Transcriptome of the Nematode Trap-Crop, Solanum sisymbriifolium
Article Snippet: .. The Iso-Seq libraries for four organs, root, stem, leaf and bud, were prepared for Isoform Sequencing (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol as described by Pacific Biosciences ( https://goo.gl/ij71Hh ) with the following modifications. .. For cDNA conversion, 3 µg of total RNA was put into each Clonetech SMARTer reaction.

Article Title: Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica *
Article Snippet: .. High yields of full-length cDNAs were generated from 1 μg total RNA of normal and fibrosis HSCs using SMART PCR cDNA synthesis kit (Clontech, USA). cDNAs (0.2 μg) were electrophosed on a 1.0% agarose gel, transferred onto Hybond N+ Membrane (Roch, Germany) and hybridized with DIG-labelled clone probes. .. These probes were generated by PCR amplification of the relevant inserts, adaptor-cutting and column-purification.

Article Title: Decreased endothelin receptor B expression in large primary uveal melanomas is associated with early clinical metastasis and short survival
Article Snippet: .. Suppression subtractive hybridisation (SSH) Double stranded cDNA was prepared from 2 μg of total RNA using the SMART™ PCR cDNA synthesis kit (Clontech, Cowley, Oxford, UK), following the manufacturer's protocol. .. SSH was performed using the PCR-Select™ cDNA subtraction kit (Clontech), again, following the manufacturer's recommended protocol.

Article Title: Identification of differentially expressed genes in normal mucosa, adenoma and adenocarcinoma of colon by SSH
Article Snippet: .. High yields of full-length cDNAs were generated from 1 μg total RNA of normal mucosa, adenoma and adenocarcinoma using SMART PCR cDNA Synthesis kit (Clontech, USA). .. 0.2 μg cDNAs were electrop hosed on a 1.0% agarose gel, transferred onto Hybond N+ Membrane (Boeheringer Mannheim, Germany) and hybridized with DIG-labeled clone probes.

Article Title: A novel method to isolate the common fraction of two DNA samples: hybrid specific amplification (HSA)
Article Snippet: .. Double-stranded cDNA was generated with the SMART PCR cDNA Synthesis Kit (Clontech). .. Aliquots of 2 µg of Qiagen-prepared BAC DNA and 1 µg of SMART cDNA PCR product were digested by Rsa I (15 U) for 90 min at 37°C.

Article Title: Cloning and expression of an inhibitor of microbial metalloproteinases from insects contributing to innate immunity
Article Snippet: .. Subtractive hybridization and suppressive PCR were carried out with the SMART PCR cDNA Synthesis Kit (Clontech) and the PCR Select cDNA Subtraction Kit (Clontech), followed by a screening with non-radioactive digoxygenin dot blots (as previously described in [ ]). .. Among the clones obtained, we identified a fragment of the IMPI cDNA.

Article Title: PR-10, defensin and cold dehydrin genes are among those over expressed in Oxytropis (Fabaceae) species adapted to the arctic
Article Snippet: .. Suppressive subtraction cDNA library construction Extracted RNA from two plantlets of a species were pooled prior to complimentary DNA (cDNA) synthesis, and 1 mg of this pool was used as template for cDNA synthesis using the SuperSmart PCR cDNA Synthesis kit (Clontech, Mountain View, CA). .. Genes specifically expressed in either the arctic or in the temperate species were isolated by applying the suppression subtractive hybridization strategy (Diatchenko et al. ) using the PCR-select cDNA subtraction kit (Clontech, Mountain View, CA).

Article Title: Long-read assays shed new light on the transcriptome complexity of a viral pathogen
Article Snippet: .. cDNA synthesisCopy DNAs were generated from polyA(+) RNA fractions using SMARTer PCR cDNA Synthesis Kits (Clontech) according to ‘PacBio Isoform Sequencing (Iso-Seq) using Clontech SMARTer PCR cDNA Synthesis Kit and No Size Selection’ protocols. .. Samples from different infection time points (1-, 4-, 8-, and 12-h p.i.) were mixed for RSII library preparation, whereas 1-, 2-, 3-, 4-, 6-, and 8-h p.i. samples were mixed for Sequel sequencing.

Hybridization:

Article Title: Decreased endothelin receptor B expression in large primary uveal melanomas is associated with early clinical metastasis and short survival
Article Snippet: .. Suppression subtractive hybridisation (SSH) Double stranded cDNA was prepared from 2 μg of total RNA using the SMART™ PCR cDNA synthesis kit (Clontech, Cowley, Oxford, UK), following the manufacturer's protocol. .. SSH was performed using the PCR-Select™ cDNA subtraction kit (Clontech), again, following the manufacturer's recommended protocol.

Article Title: Cloning and expression of an inhibitor of microbial metalloproteinases from insects contributing to innate immunity
Article Snippet: .. Subtractive hybridization and suppressive PCR were carried out with the SMART PCR cDNA Synthesis Kit (Clontech) and the PCR Select cDNA Subtraction Kit (Clontech), followed by a screening with non-radioactive digoxygenin dot blots (as previously described in [ ]). .. Among the clones obtained, we identified a fragment of the IMPI cDNA.

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    TaKaRa quantitative reverse transcription real time pcr qpcr cdna
    (a) Restriction digestion analysis of the canine <t>HRI-bacmid</t> DNA verifies the insertion of canine HRI <t>cDNA</t> into the bacmid DNA with correct orientation. Lane 1, Molecular Weight markers; Lane 2, pFastBac-canine HRI cDNA. (b) Expression of mammalian HRIs. HRI, expressed in insect cells, was detected by Western blot analysis using an affinity purified mouse monoclonal antibody against histidine and visualized by enhanced chemiluminescence. Lane 1, canine HRI; Lane 2, mouse HRI; Lane 3, rat HRI; Lane 4, human HRI.
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    Comparison between microarray data and qRT-PCR result for lncRNAs. The validation results indicated that the microarray data correlated well with the qPCR results.

    Journal: Mediators of Inflammation

    Article Title: Insights from lncRNAs Profiling of MIN6 Beta Cells Undergoing Inflammation

    doi: 10.1155/2016/9275106

    Figure Lengend Snippet: Comparison between microarray data and qRT-PCR result for lncRNAs. The validation results indicated that the microarray data correlated well with the qPCR results.

    Article Snippet: Quantitative Real-Time Reverse Transcription PCR (qRT-PCR) Reaction cDNA was synthesized using the PrimeScript™ RT Master Mix (Perfect Real Time, TAKARA, Japan) according to the manufacturer's recommendations.

    Techniques: Microarray, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Gel electrophoresis results of PCR products for full-length analysis of porcine MSRB3 cDNA. a , b and c are gel electrophoresis results for part cDNA sequences by using primers M1, M2 and M3, respectively. d and e are gel electrophoresis results of 3’-RACE and 5’-RACE. Markers in A, B and C are MarkerII(TIANGEN, China). Marks in D and E are DL2000TM DNA Marker (TaKaRa, Japan)

    Journal: Journal of Animal Science and Biotechnology

    Article Title: Porcine methionine sulfoxide reductase B3: molecular cloning, tissue-specific expression profiles, and polymorphisms associated with ear size in Sus scrofa

    doi: 10.1186/s40104-015-0060-x

    Figure Lengend Snippet: Gel electrophoresis results of PCR products for full-length analysis of porcine MSRB3 cDNA. a , b and c are gel electrophoresis results for part cDNA sequences by using primers M1, M2 and M3, respectively. d and e are gel electrophoresis results of 3’-RACE and 5’-RACE. Markers in A, B and C are MarkerII(TIANGEN, China). Marks in D and E are DL2000TM DNA Marker (TaKaRa, Japan)

    Article Snippet: Molecular cloning of full-length MSRB3 cDNA Reverse transcription was performed on MSRB3 mRNA using a PrimeScriptTM RT reagent kit (TakaRa, Japan) according to manufacturer’s instructions.

    Techniques: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Marker

    (a) Restriction digestion analysis of the canine HRI-bacmid DNA verifies the insertion of canine HRI cDNA into the bacmid DNA with correct orientation. Lane 1, Molecular Weight markers; Lane 2, pFastBac-canine HRI cDNA. (b) Expression of mammalian HRIs. HRI, expressed in insect cells, was detected by Western blot analysis using an affinity purified mouse monoclonal antibody against histidine and visualized by enhanced chemiluminescence. Lane 1, canine HRI; Lane 2, mouse HRI; Lane 3, rat HRI; Lane 4, human HRI.

    Journal: Advances in Hematology

    Article Title: Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis

    doi: 10.1155/2009/251915

    Figure Lengend Snippet: (a) Restriction digestion analysis of the canine HRI-bacmid DNA verifies the insertion of canine HRI cDNA into the bacmid DNA with correct orientation. Lane 1, Molecular Weight markers; Lane 2, pFastBac-canine HRI cDNA. (b) Expression of mammalian HRIs. HRI, expressed in insect cells, was detected by Western blot analysis using an affinity purified mouse monoclonal antibody against histidine and visualized by enhanced chemiluminescence. Lane 1, canine HRI; Lane 2, mouse HRI; Lane 3, rat HRI; Lane 4, human HRI.

    Article Snippet: Cloning of Canine HRI cDNA Reverse transcription (RT) reactions on human (Clontech, Mountain View, Calif, USA) and canine tc-RNA were performed in a 20 μ L reaction mixture containing 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 5 mM MgCl2 , 500 μ M dNTP, 1.25 μ M of oligo(dT) primer, 5 μ g tc-RNA, 40 U of RNase inhibitor, 2 U of RNaseH, and 50 U of reverse transcriptase II (Invitrogen, Carlsbad, Calif, USA).

    Techniques: Molecular Weight, Expressing, Western Blot, Affinity Purification

    (a) Expression pattern of HRI in human tissues. RT-PCR products from human tissue using HRI specific primers were separated on a 0.8% agarose gel and visualized by UV. Lanes: 1, liver; 2, kidney; 3, lymph node; 4, spleen. (b) RT-PCR products of canine HRI cDNA from spleen. 50 ng of the resulting cDNA PCR products were separated on an agarose gel (0.8%), stained with ethidium bromide and visualized by UV. (c) Nucleotide sequence comparison of the canine HRI with human, rat and mouse HRI. Conserved nucleotides between all four species are denoted with a star. (d) Amino acid sequence alignment of the canine HRI with the mouse, rat, and human HRI. Semi-conserved residues are designated with a period underneath, conserved residues are shown by semicolon underneath, and the identical residues have stars underneath them. The residues important for ATP binding are highlighted in gray. (e) Alignment of the residues involved in ATP binding. Residues important for ATP binding for the human, mouse, rat, and canine HRI are shown. Divergences between the species are highlighted.

    Journal: Advances in Hematology

    Article Title: Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis

    doi: 10.1155/2009/251915

    Figure Lengend Snippet: (a) Expression pattern of HRI in human tissues. RT-PCR products from human tissue using HRI specific primers were separated on a 0.8% agarose gel and visualized by UV. Lanes: 1, liver; 2, kidney; 3, lymph node; 4, spleen. (b) RT-PCR products of canine HRI cDNA from spleen. 50 ng of the resulting cDNA PCR products were separated on an agarose gel (0.8%), stained with ethidium bromide and visualized by UV. (c) Nucleotide sequence comparison of the canine HRI with human, rat and mouse HRI. Conserved nucleotides between all four species are denoted with a star. (d) Amino acid sequence alignment of the canine HRI with the mouse, rat, and human HRI. Semi-conserved residues are designated with a period underneath, conserved residues are shown by semicolon underneath, and the identical residues have stars underneath them. The residues important for ATP binding are highlighted in gray. (e) Alignment of the residues involved in ATP binding. Residues important for ATP binding for the human, mouse, rat, and canine HRI are shown. Divergences between the species are highlighted.

    Article Snippet: Cloning of Canine HRI cDNA Reverse transcription (RT) reactions on human (Clontech, Mountain View, Calif, USA) and canine tc-RNA were performed in a 20 μ L reaction mixture containing 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 5 mM MgCl2 , 500 μ M dNTP, 1.25 μ M of oligo(dT) primer, 5 μ g tc-RNA, 40 U of RNase inhibitor, 2 U of RNaseH, and 50 U of reverse transcriptase II (Invitrogen, Carlsbad, Calif, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Staining, Sequencing, Binding Assay