Journal: BMC Neuroscience
Article Title: VAMP-2, SNAP-25A/B and syntaxin-1 in glutamatergic and GABAergic synapses of the rat cerebellar cortex
doi: 10.1186/1471-2202-12-118
Figure Lengend Snippet: Double labelling for vGluT-1 and VAMP-2, VGluT-1 and SNAP-25A/B, VGluT-1 and syntaxin-1 . (a, b, c; g, h, i; m, n, o): molecular/Purkinje layer; (d, e, f; j, k, l; p, q, r): granular layer. In the molecular/Purkinje and granular layers, numerous punctate elements which display co-localization of vGluT-1 with VAMP-2 (c, f) or SNAP-25A/B (i, l) or syntaxin-1 (o, r) are seen. However, in all layers, a number of vGluT-1-immunoreactive puncta appear unlabelled for VAMP-2, SNAP-25A/B and syntaxin-1. Scale bars: a, b, c, d, e, f, m, n, o, p, q, r: 20 μm; g, h, i, j, k, l: 50 μm.
Article Snippet: The following commercial antibodies were used: 1. anti-vGluT-1 polyclonal antibody, raised in guinea pig against a synthetic linear peptide from rat vGluT-1 (Millipore, Billerica, MA, USA) [ , ]; 2. anti-vGluT-2 polyclonal antibody, raised in rabbit against a strep-tag fusion protein of rat vGluT-2 (Synaptic System, Goettingen, Germany) [ , ]; 3. (a) anti-GAD monoclonal antibody, raised in mouse against human GAD-65-GST fusion protein (Stressgen, Victoria, BC, Canada), and (b) anti-GAD polyclonal antibody, raised in rabbit against a synthetic peptide corresponding to the rat GAD-65 C-terminus residues 572-585 (Chemicon), which react to both GAD-65 and GAD-67 [ ]; 4. (a) anti-VAMP monoclonal antibody, raised in mouse against crude synaptic immunoprecipitate (human) (Millipore), and (b) anti-VAMP polyclonal antibody, raised in rabbit against a synthetic peptide based on rat VAMP-2 (Stressgen), which react to VAMP-2 [ ]; 5. anti-SNAP-25 monoclonal antibody, raised in against crude synaptic immunoprecipitate (human), which reacts with both SNAP-25A and SNAP-25B (Chemicon) [ ]; 6. anti-syntaxin monoclonal antibody, raised in mouse against clone HCP-1, which reacts to syntaxin-1 (ABCAM, Cambridge, UK) [ ].
Techniques:
![From the genomic sequence of the SmGLK2 gene to protein structure. (A) SmGLK2 annotated gene structure in 67/3, HQ-1315, and GUIQIE-1 eggplant reference genomes and SL4.0 tomato genome [5ʹ-untranslated region (UTR) in blue, eggplant annotated exons in yellow, tomato annotated exons in red, and 3ʹ-UTR in green]. (B) The S. melongena 67/3 (SRR3884608), S. incanum INC1 (SRR2289250), and S. insanum MM0686 accession (SRR8736646) transcripts aligned against the SmGLK2 gene sequence from the 67/3 v.3 eggplant reference genome and visualized in the IGV tool ( Robinson et al. , 2023 ). The candidate structural variation identified is indicated with a purple line. (C) The suggested SmGLK2 gene structural annotation as a consensus of previous information with the extra proposed exon in purple. The identified structural variation in the gene sequence is indicated with a black arrowhead and a red line. (D) Comparison of mRNA sequences for presence (FN) or absence (fn) of the fruit green netting trait. The premature stop codon downstream of the indel is indicated with an asterisk. (E) From left to right: comparison of the protein structure and sequence around the indel site for FN and fn with the nuclear localization signal (NLS) indicated in dark grey and the golden2-like transcription factor domain in green; western blot showing the difference in apparent molecular mass of the <t>GLK2</t> protein from FN and fn tissues; and differences in the expression level of GLK1 and GLK2 from different tissues of S. insanum INS1. PP2AA2 gene was used as a reference gene for normalization. Columns and error bars represent means and standard deviations of three replicates. Different letters above the columns indicate statistical significance ( P <0.05) according to Student’s t -test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0072/pmc11630072/pmc11630072__erae355_fig2.jpg)
