Journal: PLoS ONE

Article Title: Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development

doi: 10.1371/journal.pone.0129944

Figure Lengend Snippet: (A-B) RT-PCR analyses for exons encoding the amino-terminus of Kidins220 and between exons 24 and 30 (24f-30r) were carried out on adult mouse (A) and human (B) tissue panels. Tissues are labelled by capital letters. N indicates PCR products obtained using primers designed to recognise exons 3 and 8 in mouse, and exon 9 and 13 in human. 24f-30r indicates samples obtained by amplification with primers recognising exons 24 and 30. Arrowheads point to samples in which a specific alternative splicing pattern was detected. (C-D) Schematics of Kidins220 splice isoforms identified in mouse heart and brain (C) and human brain (D) in the region encoded by exons 24 to 30.

Article Snippet: PC12 cells were transfected using a Polyplus JetPEI transfection kit (using 0.5 μg pLVX Tet-ON vector and 0.5 μg of the respective Kidins220 isoform construct) and were differentiated with 100 ng/ml NGF (Alomone) for 48 h. HEK cells stably transfected with full-length TrkA [ ] were grown in DMEM containing 10% foetal bovine serum and 4 mM glutamine at 37°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: PLoS ONE

Article Title: Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development

doi: 10.1371/journal.pone.0129944

Figure Lengend Snippet: (A-B) RT-PCR analysis for the carboxy-terminal endings of Kidins220 (exon 32 and exon 33) was carried out on an adult mouse (A) and human (B) tissue panels. Capital letters indicate the different tissues. 31f and 33 rev indicate amplification with primers against exons 31 and 33. Arrowheads point out samples where alternative terminal exon (ATE) splicing for Kidins220 is detected. (C) PCR products obtained using primers designed to recognise exons encoding the carboxy-terminus of Kidins220 (exons 31/32 and 32 in mouse and exon 31 and 32 in human).

Article Snippet: PC12 cells were transfected using a Polyplus JetPEI transfection kit (using 0.5 μg pLVX Tet-ON vector and 0.5 μg of the respective Kidins220 isoform construct) and were differentiated with 100 ng/ml NGF (Alomone) for 48 h. HEK cells stably transfected with full-length TrkA [ ] were grown in DMEM containing 10% foetal bovine serum and 4 mM glutamine at 37°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: PLoS ONE

Article Title: Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development

doi: 10.1371/journal.pone.0129944

Figure Lengend Snippet: Western blots of embryonic day 18.5 (E) and adult (A) brain lysates show evidence of protein translation of Kidins220 variants containing: amino-terminus (N-term), Kidins220 isoform 1 (m1), Kidins220 isoform 6 (m6) and exon 33 (C2). Actin was used a loading control.

Article Snippet: PC12 cells were transfected using a Polyplus JetPEI transfection kit (using 0.5 μg pLVX Tet-ON vector and 0.5 μg of the respective Kidins220 isoform construct) and were differentiated with 100 ng/ml NGF (Alomone) for 48 h. HEK cells stably transfected with full-length TrkA [ ] were grown in DMEM containing 10% foetal bovine serum and 4 mM glutamine at 37°C.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development

doi: 10.1371/journal.pone.0129944

Figure Lengend Snippet: (A-B) RT-PCR analyses for exons encoding the amino-terminus of Kidins220 and between exons 24 and 30 (A), and for alternative terminal exon splicing (B) were carried out on mouse brains at different developmental stages, starting from embryonic stage E13.5 up to postnatal stage P64. The analysis at the latter stage includes adult male (M) and female (F) brains. N indicates PCR products obtained using primers designed to recognise exons 3 and 8. 24f-30r indicates samples obtained by amplification with primers recognising exons 24 and 30 (A). C indicates PCR products obtained using primers designed to recognise exons 31/32 and 32. 31f-33r indicates samples obtained by amplification with primers recognising exons 31 and 33 (B). The arrowhead points to appearance of Kidins220 isoform m1 (full-length) at postnatal stage P11 (A).

Article Snippet: PC12 cells were transfected using a Polyplus JetPEI transfection kit (using 0.5 μg pLVX Tet-ON vector and 0.5 μg of the respective Kidins220 isoform construct) and were differentiated with 100 ng/ml NGF (Alomone) for 48 h. HEK cells stably transfected with full-length TrkA [ ] were grown in DMEM containing 10% foetal bovine serum and 4 mM glutamine at 37°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: PLoS ONE

Article Title: Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development

doi: 10.1371/journal.pone.0129944

Figure Lengend Snippet: (A-F) RNA extracted from cortical (A, D), hippocampal (B, E) and motor neuron (C, F) primary cultures was reverse transcribed into cDNA. N indicates PCR products obtained using primers designed to recognise exons 3 and 8 of Kidins220. 24f-30r indicates samples obtained by amplification with primers recognising exons 24 and 30 (A-C). C indicates PCR products obtained using primers designed to recognise exons 31/32 and 32. 31f-33r indicates samples obtained by amplification with primers recognising exons 31 and 33 (D-F). Note the absence of ATE splicing isoforms C1 and C2 in primary motor neuron cultures.

Article Snippet: PC12 cells were transfected using a Polyplus JetPEI transfection kit (using 0.5 μg pLVX Tet-ON vector and 0.5 μg of the respective Kidins220 isoform construct) and were differentiated with 100 ng/ml NGF (Alomone) for 48 h. HEK cells stably transfected with full-length TrkA [ ] were grown in DMEM containing 10% foetal bovine serum and 4 mM glutamine at 37°C.

Techniques: Amplification

Journal: PLoS ONE

Article Title: Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development

doi: 10.1371/journal.pone.0129944

Figure Lengend Snippet: (A-B) Primary cortical (A) and hippocampal (B) neurons were prepared from E18.5 mouse embryos and half of each culture was plated with medium containing 100 ng/ml BDNF. RNA was extracted at different time points and reverse transcribed. N indicates PCR products obtained using primers designed to recognise exons 3 and 8. 24f-30r indicates samples obtained by amplification with primers recognising exons 24 and 30. (A) Arrowheads point to early appearance of Kidins220 isoform m1 (full-length) in BDNF treated cultures compared to non-treated cultures.

Article Snippet: PC12 cells were transfected using a Polyplus JetPEI transfection kit (using 0.5 μg pLVX Tet-ON vector and 0.5 μg of the respective Kidins220 isoform construct) and were differentiated with 100 ng/ml NGF (Alomone) for 48 h. HEK cells stably transfected with full-length TrkA [ ] were grown in DMEM containing 10% foetal bovine serum and 4 mM glutamine at 37°C.

Techniques: Amplification

Journal: PLoS ONE

Article Title: Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development

doi: 10.1371/journal.pone.0129944

Figure Lengend Snippet: (A) Schematics of Kidins220 splice isoform m6 and Kidins220 ATE m6/C2 (from Exon 24 onwards) used for transfection of PC12 cells in Fig 7B. (B) PC12 cells were transfected with HA-tagged Kidins220 isoform m6, isoform m6/C2 or with Tet-ON pLVX vector only (control) and after 6 h stimulated with doxycycline and differentiated for 48 h with NGF. Full-length Kidins220 was detected using a polyclonal antibody directed against the carboxy-terminus of Kidins220 (GSC16 antibody; in green). An anti-HA antibody was used to stain Kidins220 isoforms m6 and m6/C2 (in red). The gain of the red channel was enhanced equally for cells overexpressing isoform m6/C2 and control cells, whilst it was tuned down for PC12 cells transfected with isoform m6 to adjust for the higher expression levels of this Kidins220 variant. Boxed areas of the merged images are magnified on the right. Representative pictures were chosen from three different experiments. Scale bars, 10 μm.

Article Snippet: PC12 cells were transfected using a Polyplus JetPEI transfection kit (using 0.5 μg pLVX Tet-ON vector and 0.5 μg of the respective Kidins220 isoform construct) and were differentiated with 100 ng/ml NGF (Alomone) for 48 h. HEK cells stably transfected with full-length TrkA [ ] were grown in DMEM containing 10% foetal bovine serum and 4 mM glutamine at 37°C.

Techniques: Transfection, Plasmid Preparation, Staining, Expressing, Variant Assay

Journal: PLoS ONE

Article Title: Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development

doi: 10.1371/journal.pone.0129944

Figure Lengend Snippet: PC12 cells were transfected with HA-tagged Kidins220 isoform m6, isoform m6/C2 or with Tet-ON pLVX vector only (control) and after 6 h stimulated with doxycycline for 48 h. Samples were stained for TrkA (in green) and for HA (in red). Arrowheads indicate transfected cells. All channels were adjusted equally to accommodate the increase in TrkA levels upon m6/C2 expression. Representative pictures were chosen from three different experiments. Scale bars, 10 μm.

Article Snippet: PC12 cells were transfected using a Polyplus JetPEI transfection kit (using 0.5 μg pLVX Tet-ON vector and 0.5 μg of the respective Kidins220 isoform construct) and were differentiated with 100 ng/ml NGF (Alomone) for 48 h. HEK cells stably transfected with full-length TrkA [ ] were grown in DMEM containing 10% foetal bovine serum and 4 mM glutamine at 37°C.

Techniques: Transfection, Plasmid Preparation, Staining, Expressing