Journal: Frontiers in Neurology

Article Title: A novel truncating variant of SPAST associated with hereditary spastic paraplegia indicates a haploinsufficiency pathogenic mechanism

doi: 10.3389/fneur.2022.1005544

Figure Lengend Snippet: Schematic structure of the human wild-type and mutant spastin proteins. M1 and M87 spastin isoforms are presented. HD, hydrophobic domain; MIT, microtubule interacting and transport domain; MBD, microtubule-binding domain; AAA ATPase, ATPase associated with diverse cellular activities domain. The black arrow indicates the location of the c.862_863dupAC frameshift variant.

Article Snippet: After SDS-PAGE, proteins were transferred onto the PVDF membrane and then immunoblotted using an anti-spastin rabbit polyclonal antibody (#PA5-53581), which could recognize both the M1 and M87 spastin isoforms (Immunogen sequence region: amino acids 234-366).

Techniques: Mutagenesis, Binding Assay, Variant Assay

Journal: Frontiers in Neurology

Article Title: A novel truncating variant of SPAST associated with hereditary spastic paraplegia indicates a haploinsufficiency pathogenic mechanism

doi: 10.3389/fneur.2022.1005544

Figure Lengend Snippet: Transcription analysis of spastin in lymphoblast cell lines. (A) Agarose gel electrophoresis of the long-range RT-PCR products revealed only the cDNA fragment of 1,494 bp corresponding to the predicted canonical transcripts in both the patient and the control. (B) Sanger sequencing of long-range RT-PCR products revealed only the canonical transcript in the proband, which was reconfirmed on sequencing with a reverse primer. The transcript with the frameshift variant was not detected. (C) Agarose gel electrophoresis of the shorter RT-PCR products revealed only the 603 bp band expected in both the patient and control. (D) Sanger sequencing of the shorter RT-PCR products in both directions identified only canonical transcripts in the patient. cDNA, complementary DNA; gDNA, genomic DNA.

Article Snippet: After SDS-PAGE, proteins were transferred onto the PVDF membrane and then immunoblotted using an anti-spastin rabbit polyclonal antibody (#PA5-53581), which could recognize both the M1 and M87 spastin isoforms (Immunogen sequence region: amino acids 234-366).

Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Control, Sequencing, Variant Assay

Journal: Frontiers in Neurology

Article Title: A novel truncating variant of SPAST associated with hereditary spastic paraplegia indicates a haploinsufficiency pathogenic mechanism

doi: 10.3389/fneur.2022.1005544

Figure Lengend Snippet: Expression analysis of spastin in lymphoblast cell lines. (A) Quantitative PCR of SPAST cRNA showed a ~50% decrease standardized against 18SRNA in the proband compared with 6 wild-type controls. Data were the mean (±SD) of three independent experiments, Mann-Whitney U test; * p < 0.01. (B) Western Blotting analysis revealed a decreased level of the spastin protein in the patient compared to the control. Blots are representative images from 3 independent experiments. (C) Densitometry analysis of the peak density normalized over β-actin showed a ~40% decrease of spastin protein in the patient compared with the control. Data were the mean (±SD) of three independent experiments, * P < 0.05. The Mann-Whitney test was used to compare the two groups.

Article Snippet: After SDS-PAGE, proteins were transferred onto the PVDF membrane and then immunoblotted using an anti-spastin rabbit polyclonal antibody (#PA5-53581), which could recognize both the M1 and M87 spastin isoforms (Immunogen sequence region: amino acids 234-366).

Techniques: Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Western Blot, Control

Journal:

Article Title: Modulation of paratrigeminal nociceptive neurons following temporomandibular joint inflammation in rats

doi: 10.1016/j.expneurol.2008.08.005

Figure Lengend Snippet: Fos-LI cells in the Pa5 after CFA or saline injection into the TMJ. Fluorogold injection site (A), retrogradely labeled FG and Fos/FG positive cells (B, C. D, E and F) and the mean number of Fos-LI cells (G) ipsilateral side to CFA injection and (I) contralateral side to CFA injection), Fos/FG-LI cells in the ipsilateral side to CFA injection (H). A: FG injection site indicated by the arrow. B: Pa5 with Fos-LI cells and Fos/FG-LI cells in the ipsilateral side to TMJ CFA injection. C: Fos-LI cells in the ipsilateral side to TMJ CFA injection as indicated by the arrows. D: Fos/FG-LI cells in the ipsilateral side to TMJ CFA injection as indicated by the arrows. E: A FG-LI cell in the ipsilateral side to TMJ CFA injection. F: Fos-LI cells in the contralateral side to TMJ CFA injection. G: Mean number of Fos-LI cells in the ipsilateral side to TMJ CFA injection. H: Mean number of FG/Fos-LI cells in the ipsilateral side to TMJ CFA injection. I: Mean number of Fos-LI cells in the contralateral side to TMJ CFA injection. PBNL: parabrachial nucleus lateralis, PSV: trigeminal spinal nucleus principalis. *: p<0.05(vs. TMJ-untreated or TMJ-vehicle, as indicated).

Article Snippet: Indeed, the Pa5 appears to have a complex role in nocifensive behavior ( Koepp et al., 2006 ).

Techniques: Saline, Injection, Labeling

Journal:

Article Title: Modulation of paratrigeminal nociceptive neurons following temporomandibular joint inflammation in rats

doi: 10.1016/j.expneurol.2008.08.005

Figure Lengend Snippet: Location of the recording sites in Pa5 ipsilateral to the CFA injection side. (A) A photomicrograph of the Pa5 with a lesion indicated by an arrow, (B) the camera lucida drawings of recording sites of WDR and NS neurons in Pa5.

Article Snippet: Indeed, the Pa5 appears to have a complex role in nocifensive behavior ( Koepp et al., 2006 ).

Techniques: Injection