Journal: BMC Neuroscience
Article Title: Stable silencing of SNAP-25 in PC12 cells by RNA interference
doi: 10.1186/1471-2202-7-9
Figure Lengend Snippet: Specific silencing of SNAP-25 by RNA interference . A) Immunoblots were done to assess the levels of SNAP-25, SNAP-23, synaptotagmin I, syntaxin 1A, tyrosine hydroxylase and β-actin in wild type PC12 cells, SNAP-25 knockdown cells and in control transfected cells (PC12 cells stably transfected with pG418-shRNA lacking an shRNA insert). Equal amounts of protein were loaded per lane. B) The SNAP-25 phenotype is maintained for at least 10 weeks in culture in both the parent PC12 cell line and the SNAP-25 knockdown cell line. C) Human and zebrafish SNAP-25 mRNAs are resistant to RNA interference. The specificity of the SNAP-25 shRNA was demonstrated by transiently transfecting SNAP-25 knockdown cells with plasmids designed to express SNAP-25 cDNA of rat, human, or zebrafish origin. The 19 nucleotide region of rat SNAP-25 mRNA which is targeted by the SNAP-25 shRNA differs from human SNAP-25 RNA at only 2 positions and from zebrafish SNAP-25 mRNA in 4 positions. The immunoblot shows that expression of rat SNAP-25 was silenced in the SNAP-25 knockdown cells, but human and zebrafish SNAP-25 were expressed. All three SNAP-25 cDNAs appeared to be expressed in the control transfected cells in that there was more SNAP-25 in the control transfected cells than in untransfected PC12 cells. The immunoblot was stripped and reprobed for β-actin to demonstrate approximately equal amounts of protein in each sample. D) SNAP-25 mRNA is reduced in SNAP-25 knockdown cells. RT-PCR was carried out with RNA isolated from wild type PC12 cells, SNAP-25 knockdown cells and in control transfected cells. SNAP-25 mRNA was easily detected in wild type and control transfected PC12 cells, but no SNAP-25 mRNA was detected in the SNAP-25 knockdown cells in this PCR experiments. However, if more of the reverse-transcription product was used for the PCR or if more cycles were done in the PCR reaction, some SNAP-25 mRNA was detectable in the SNAP-25 knockdown cells.
Article Snippet: Levels of SNAP-25, SNAP-23, syntaxin 1A, synaptotagmin I, tyrosine hydroxylase and β-actin in the cell lines were assessed by immunoblotting with the following antibodies: SNAP-25 (ANR-001, Alomone), SNAP-23 (DS-19; Sigma), syntaxin 1A (#573831, Calbiochem), synaptotagmin I (mAb48; Developmental Systems Hybridoma Bank, University of Iowa), tyrosine hydroxylase (#657010, Calbiochem), β-actin (JLA20; Developmental Systems Hybridoma Bank, University of Iowa), and horseradish peroxidase-labeled anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch).
Techniques: Western Blot, Transfection, Stable Transfection, shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation
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![Increased Immunolabeling Efficiency in Epoxy-Resin-Embedded Tissue after Etching and Antigen Retrieval (A and B) Post-embedding fluorescent labeling for <t>SNAP25</t> on 200-nm-thin sections obtained from rat cerebellar cortex embedded into three different types of resin without (A) or with 2 min of etching with Na-ethanolate (B). All images were taken under the same illumination and detection conditions. (C) Munc13-1 immunolabeling of the stratum radiatum in epoxy-resin-embedded mouse hippocampal CA1 area after etching and retrieval. Reactions were carried out on sections with different thicknesses. (D) Normalized mean fluorescent signal intensity of PSD95, Munc13-1, and vGluT1 show a tight linear correlation with the section thickness. Symbols for each protein are the mean ± SD from 3 reactions in 3 mice. The reaction strengths were normalized to those obtained in the 200-nm-thick sections. (E) Immunofluorescence for vGluT1 in the stratum radiatum of the CA1 area (left image). Circular ROIs were placed over fluorescent clusters (ROI #1–#4) and over the unlabeled neuropil to determine the specific and background (bg) labeling, respectively. The reaction was followed by an elution step with 1% SDS (middle image) and a step when only the appropriate sAb (Alexa647 coupled donkey anti-rabbit) was applied (right image). All images were taken with the same acquisition settings and are shown with the same look-up table. (F) The integrated fluorescence (background-subtracted mean) is plotted for the 1 st labeling round, the elution, and the sAb relabeling steps. When the sAb was reapplied, the mean fluorescence did not increase significantly (1.4% ± 0.5% versus 2.3% ± 1.1% of the 1 st labeling round, n = 7, p = 0.99; while both significantly differed from the 1 st labeling round, p < 0.0002, Kruskal-Wallis with Tukey honestly significant difference [HSD] post hoc test). Measurements for 7 proteins are shown, and symbols for each protein are the mean ± SD from 3 reactions in 3 mice from 20–50 ROIs per reaction. (G) Mean normalized PSD95 fluorescence is shown in 4 consecutive labeling rounds in epoxy-resin-embedded tissue obtained from 3 mice (open circles, 60, 69, and 73 ROIs in 3 reactions). The mean ± SD are shown with filled circles. (H) Same as in (G), but labeling for 4 proteins are shown (all represent the mean ± SD of 3 mice; 20–73 ROIs in each reaction). (I) The background-subtracted fluorescence of each ROI (same as in H) was calculated and normalized for the mean of each labeling round. Then, the ratio of the 2 nd and 1 st rounds was calculated for each ROI, and the mean and the coefficient of variation (CV) was calculated of these ratios. This was repeated for the 3 rd and 2 nd rounds and for the 4 th and 3 rd rounds. The symbols represent the mean ± SD from 3 reactions (3 mice). (J) The ratios of the background-subtracted fluorescent signal for vGluT1 and PSD95 and for Munc13-1 and PSD95 were calculated across 4 labeling rounds (27–30 ROIs in each experiment in 3 mice). Then, the ratios of the 2 nd and 1 st , 3 rd and 2 nd , and 4 th and 3 rd rounds were calculated. The round-to-round variability (CV) was then calculated and plotted. Open symbols represent the CV values of the 3 reactions in 3 mice, whereas filled symbols indicate the mean ± SD. Abbreviations are as follows: ml, molecular layer; pcl, Purkinje cell layer; gcl, granular layer.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8500/pmc07408500/pmc07408500__gr1.jpg)
