snap 25  (Alomone Labs)


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    Structured Review

    Alomone Labs snap 25
    Plasmid-based expression of <t>shRNA-SNAP-25</t> . A) Plasmid map of pG418-shRNA, a plasmid designed to express shRNAs from the mouse U6 promoter. B) The two 56-base deoxyoligonucleotides used in the construction of pG418-shRNA-SNAP-25 are shown as they would be paired after annealing. The labels above and below the oligonucleotides indicate the source or the function of the nucleotides in the indicated regions. C) The predicted stem-loop structure of shRNA-SNAP-25 expressed from pG418-shRNA-SNAP-25.
    Snap 25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/snap 25/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    snap 25 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Stable silencing of SNAP-25 in PC12 cells by RNA interference"

    Article Title: Stable silencing of SNAP-25 in PC12 cells by RNA interference

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-7-9

    Plasmid-based expression of shRNA-SNAP-25 . A) Plasmid map of pG418-shRNA, a plasmid designed to express shRNAs from the mouse U6 promoter. B) The two 56-base deoxyoligonucleotides used in the construction of pG418-shRNA-SNAP-25 are shown as they would be paired after annealing. The labels above and below the oligonucleotides indicate the source or the function of the nucleotides in the indicated regions. C) The predicted stem-loop structure of shRNA-SNAP-25 expressed from pG418-shRNA-SNAP-25.
    Figure Legend Snippet: Plasmid-based expression of shRNA-SNAP-25 . A) Plasmid map of pG418-shRNA, a plasmid designed to express shRNAs from the mouse U6 promoter. B) The two 56-base deoxyoligonucleotides used in the construction of pG418-shRNA-SNAP-25 are shown as they would be paired after annealing. The labels above and below the oligonucleotides indicate the source or the function of the nucleotides in the indicated regions. C) The predicted stem-loop structure of shRNA-SNAP-25 expressed from pG418-shRNA-SNAP-25.

    Techniques Used: Plasmid Preparation, Expressing, shRNA

    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.
    Figure Legend 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.

    Techniques Used: Western Blot, Transfection, Stable Transfection, shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation

    Catecholamine secretion is reduced in SNAP-25 knockdown PC12 cells . Representative amperometric traces are shown from a control cell stably transfected with pG418-shRNA lacking an shRNA insert (empty vector) (A) and from a SNAP-25 knockdown cell prior to and during a 2.5 min stimulation with 60 mM KCl (B). To the right of each of the traces is an averaged amperometric event shown on an expanded time scale. C) When compared to empty vector controls (n = 18) the silencing of SNAP-25 in PC12 cells (n = 18) did not alter the number of norepinephrine molecules per release event. D) Silencing of SNAP-25 resulted in a 63% reduction in the total number of exocytotic events produced following stimulation. Control transfected cells and SNAP-25 knockdown cells produced a mean ± SEM of 79 ± 17 events and 29 ± 10 events, respectively. *p < 0.03 (Student's t-test). E) Transient transfection with a human SNAP-25 expression plasmid rescued the deficit in catecholamine secretion in the SNAP-25 knockdown cells. *p < 0.03 (Student's t-test).
    Figure Legend Snippet: Catecholamine secretion is reduced in SNAP-25 knockdown PC12 cells . Representative amperometric traces are shown from a control cell stably transfected with pG418-shRNA lacking an shRNA insert (empty vector) (A) and from a SNAP-25 knockdown cell prior to and during a 2.5 min stimulation with 60 mM KCl (B). To the right of each of the traces is an averaged amperometric event shown on an expanded time scale. C) When compared to empty vector controls (n = 18) the silencing of SNAP-25 in PC12 cells (n = 18) did not alter the number of norepinephrine molecules per release event. D) Silencing of SNAP-25 resulted in a 63% reduction in the total number of exocytotic events produced following stimulation. Control transfected cells and SNAP-25 knockdown cells produced a mean ± SEM of 79 ± 17 events and 29 ± 10 events, respectively. *p < 0.03 (Student's t-test). E) Transient transfection with a human SNAP-25 expression plasmid rescued the deficit in catecholamine secretion in the SNAP-25 knockdown cells. *p < 0.03 (Student's t-test).

    Techniques Used: Stable Transfection, Transfection, shRNA, Plasmid Preparation, Produced, Expressing

    snap 25  (Alomone Labs)


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    Structured Review

    Alomone Labs snap 25
    Plasmid-based expression of <t>shRNA-SNAP-25</t> . A) Plasmid map of pG418-shRNA, a plasmid designed to express shRNAs from the mouse U6 promoter. B) The two 56-base deoxyoligonucleotides used in the construction of pG418-shRNA-SNAP-25 are shown as they would be paired after annealing. The labels above and below the oligonucleotides indicate the source or the function of the nucleotides in the indicated regions. C) The predicted stem-loop structure of shRNA-SNAP-25 expressed from pG418-shRNA-SNAP-25.
    Snap 25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/snap 25/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    snap 25 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Stable silencing of SNAP-25 in PC12 cells by RNA interference"

    Article Title: Stable silencing of SNAP-25 in PC12 cells by RNA interference

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-7-9

    Plasmid-based expression of shRNA-SNAP-25 . A) Plasmid map of pG418-shRNA, a plasmid designed to express shRNAs from the mouse U6 promoter. B) The two 56-base deoxyoligonucleotides used in the construction of pG418-shRNA-SNAP-25 are shown as they would be paired after annealing. The labels above and below the oligonucleotides indicate the source or the function of the nucleotides in the indicated regions. C) The predicted stem-loop structure of shRNA-SNAP-25 expressed from pG418-shRNA-SNAP-25.
    Figure Legend Snippet: Plasmid-based expression of shRNA-SNAP-25 . A) Plasmid map of pG418-shRNA, a plasmid designed to express shRNAs from the mouse U6 promoter. B) The two 56-base deoxyoligonucleotides used in the construction of pG418-shRNA-SNAP-25 are shown as they would be paired after annealing. The labels above and below the oligonucleotides indicate the source or the function of the nucleotides in the indicated regions. C) The predicted stem-loop structure of shRNA-SNAP-25 expressed from pG418-shRNA-SNAP-25.

    Techniques Used: Plasmid Preparation, Expressing, shRNA

    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.
    Figure Legend 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.

    Techniques Used: Western Blot, Transfection, Stable Transfection, shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation

    Catecholamine secretion is reduced in SNAP-25 knockdown PC12 cells . Representative amperometric traces are shown from a control cell stably transfected with pG418-shRNA lacking an shRNA insert (empty vector) (A) and from a SNAP-25 knockdown cell prior to and during a 2.5 min stimulation with 60 mM KCl (B). To the right of each of the traces is an averaged amperometric event shown on an expanded time scale. C) When compared to empty vector controls (n = 18) the silencing of SNAP-25 in PC12 cells (n = 18) did not alter the number of norepinephrine molecules per release event. D) Silencing of SNAP-25 resulted in a 63% reduction in the total number of exocytotic events produced following stimulation. Control transfected cells and SNAP-25 knockdown cells produced a mean ± SEM of 79 ± 17 events and 29 ± 10 events, respectively. *p < 0.03 (Student's t-test). E) Transient transfection with a human SNAP-25 expression plasmid rescued the deficit in catecholamine secretion in the SNAP-25 knockdown cells. *p < 0.03 (Student's t-test).
    Figure Legend Snippet: Catecholamine secretion is reduced in SNAP-25 knockdown PC12 cells . Representative amperometric traces are shown from a control cell stably transfected with pG418-shRNA lacking an shRNA insert (empty vector) (A) and from a SNAP-25 knockdown cell prior to and during a 2.5 min stimulation with 60 mM KCl (B). To the right of each of the traces is an averaged amperometric event shown on an expanded time scale. C) When compared to empty vector controls (n = 18) the silencing of SNAP-25 in PC12 cells (n = 18) did not alter the number of norepinephrine molecules per release event. D) Silencing of SNAP-25 resulted in a 63% reduction in the total number of exocytotic events produced following stimulation. Control transfected cells and SNAP-25 knockdown cells produced a mean ± SEM of 79 ± 17 events and 29 ± 10 events, respectively. *p < 0.03 (Student's t-test). E) Transient transfection with a human SNAP-25 expression plasmid rescued the deficit in catecholamine secretion in the SNAP-25 knockdown cells. *p < 0.03 (Student's t-test).

    Techniques Used: Stable Transfection, Transfection, shRNA, Plasmid Preparation, Produced, Expressing

    snap 25  (Alomone Labs)


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    Alomone Labs snap 25
    (A) GFP reporter constructs were created by fusing the reading frame of GFP to the snap-25a 3′UTR. Two predicted miRNA recognition elements (MREs) were identified in the snap-25a 3′ UTR. The miR-153 sequence is indicated in red and the corresponding snap-25a UTR sequence is shown in green. (B) Single cell zebrafish embryos were injected with mRNAs derived from GFP reporters lacking a UTR (GFP), fused to the full length snap-25a UTR ( <t>+snap-25),</t> or mutant versions of the snap-25a UTR lacking individual MREs ( snap-25a ΔMRE1 and snap-25a ΔMRE2) or both MREs ( snap-25a ΔMRE1&2). Embryos were injected in the presence or absence of exogenous miR-153 or morpholinos against miR-153 ( miR-153 MO ). Fluorescence levels were examined at 1 dpf. Clusters of embryos (∼60) are shown as well as a high magnification image of a single representative embryo. (C) Lysates from ∼100 embryos were prepared from embryos treated as in B and GFP protein levels were determined by western blotting using antibodies against GFP or control antibodies against α-tubulin. (D) Quantitation of westerns was performed with a paired Student’s t-test (n = 5).
    Snap 25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/snap 25/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    snap 25 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "miR-153 Regulates SNAP-25, Synaptic Transmission, and Neuronal Development"

    Article Title: miR-153 Regulates SNAP-25, Synaptic Transmission, and Neuronal Development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057080

    (A) GFP reporter constructs were created by fusing the reading frame of GFP to the snap-25a 3′UTR. Two predicted miRNA recognition elements (MREs) were identified in the snap-25a 3′ UTR. The miR-153 sequence is indicated in red and the corresponding snap-25a UTR sequence is shown in green. (B) Single cell zebrafish embryos were injected with mRNAs derived from GFP reporters lacking a UTR (GFP), fused to the full length snap-25a UTR ( +snap-25), or mutant versions of the snap-25a UTR lacking individual MREs ( snap-25a ΔMRE1 and snap-25a ΔMRE2) or both MREs ( snap-25a ΔMRE1&2). Embryos were injected in the presence or absence of exogenous miR-153 or morpholinos against miR-153 ( miR-153 MO ). Fluorescence levels were examined at 1 dpf. Clusters of embryos (∼60) are shown as well as a high magnification image of a single representative embryo. (C) Lysates from ∼100 embryos were prepared from embryos treated as in B and GFP protein levels were determined by western blotting using antibodies against GFP or control antibodies against α-tubulin. (D) Quantitation of westerns was performed with a paired Student’s t-test (n = 5).
    Figure Legend Snippet: (A) GFP reporter constructs were created by fusing the reading frame of GFP to the snap-25a 3′UTR. Two predicted miRNA recognition elements (MREs) were identified in the snap-25a 3′ UTR. The miR-153 sequence is indicated in red and the corresponding snap-25a UTR sequence is shown in green. (B) Single cell zebrafish embryos were injected with mRNAs derived from GFP reporters lacking a UTR (GFP), fused to the full length snap-25a UTR ( +snap-25), or mutant versions of the snap-25a UTR lacking individual MREs ( snap-25a ΔMRE1 and snap-25a ΔMRE2) or both MREs ( snap-25a ΔMRE1&2). Embryos were injected in the presence or absence of exogenous miR-153 or morpholinos against miR-153 ( miR-153 MO ). Fluorescence levels were examined at 1 dpf. Clusters of embryos (∼60) are shown as well as a high magnification image of a single representative embryo. (C) Lysates from ∼100 embryos were prepared from embryos treated as in B and GFP protein levels were determined by western blotting using antibodies against GFP or control antibodies against α-tubulin. (D) Quantitation of westerns was performed with a paired Student’s t-test (n = 5).

    Techniques Used: Construct, Sequencing, Injection, Derivative Assay, Mutagenesis, Fluorescence, Western Blot, Quantitation Assay

    (A) Embryo lysates were prepared from either NIC embryos or embryos injected with miR-153 , miR-153 MO , mRNAs encoding snap-25a and snap-25b , morpholinos against snap-25, or combinations thereof, as indicated. Western blots were performed using antibodies against SNAP-25 and α–tubulin. (B) Quantification of SNAP-25 levels from the western blots (n = 3) shown in A. Significance was determined by a two-tailed Student’s t-test. Error bars show s.e.m.
    Figure Legend Snippet: (A) Embryo lysates were prepared from either NIC embryos or embryos injected with miR-153 , miR-153 MO , mRNAs encoding snap-25a and snap-25b , morpholinos against snap-25, or combinations thereof, as indicated. Western blots were performed using antibodies against SNAP-25 and α–tubulin. (B) Quantification of SNAP-25 levels from the western blots (n = 3) shown in A. Significance was determined by a two-tailed Student’s t-test. Error bars show s.e.m.

    Techniques Used: Injection, Western Blot, Two Tailed Test

    (A) Single cell embryos were injected as indicated and then at 27 hpf, exposed to Botulinum neurotoxin A (BoNT) for 30 minutes. After recovery for 1 hour, western blots were performed on embryo lysates using antibodies against SNAP-25 or α–tubulin. (B) Quantitation of SNAP-25 levels from A, n = 3. **, p<0.01 (C) Embryonic movement in the presence or absence of BoNT A. The number of twitches per minute was counted as in Fig. 1 for embryos treated as indicated. Significance was determined by comparing mock embryos to all other conditions using ANOVA with Dunnett’s post-test, n = 15. *, p<0.05.
    Figure Legend Snippet: (A) Single cell embryos were injected as indicated and then at 27 hpf, exposed to Botulinum neurotoxin A (BoNT) for 30 minutes. After recovery for 1 hour, western blots were performed on embryo lysates using antibodies against SNAP-25 or α–tubulin. (B) Quantitation of SNAP-25 levels from A, n = 3. **, p<0.01 (C) Embryonic movement in the presence or absence of BoNT A. The number of twitches per minute was counted as in Fig. 1 for embryos treated as indicated. Significance was determined by comparing mock embryos to all other conditions using ANOVA with Dunnett’s post-test, n = 15. *, p<0.05.

    Techniques Used: Injection, Western Blot, Quantitation Assay

    (A) A transgenic zebrafish line, Tg(mnx1:TagRFP-T) , that expresses RFP in motor neurons was used to monitor the effects of altered levels of miR-153 and snap-25 at 55 hpf. For all confocal images, developing motor neurons were examined from the same somites, as indicated. (B) Morphology of developing motor neurons under each of the indicated conditions. Arrows indicate increased branching after knockdown miR-153 ( miR-153 MO ) or overexpression snap-25a,b mRNA. Arrowheads indicate the structural defects after miR-153 overexpression or knockdown of snap-25a,b ( snap-25a,b MO ). Scale bar: 20 µm. (C) Quantification of motor neuron axonal branch number under the different conditions shown in (B). Error bars show s.e.m. Significance was determined using ANOVA with Dunnett’s post-test, n = 5. *, p<0.01; **, p<0.005. (D) Quantification of motor neuron axon length relative to uninjected control under the different conditions shown in (B). Error bars show s.e.m. ANOVA with Dunnett’s post-test, n = 5. *, p<0.05; **, p<0.01.
    Figure Legend Snippet: (A) A transgenic zebrafish line, Tg(mnx1:TagRFP-T) , that expresses RFP in motor neurons was used to monitor the effects of altered levels of miR-153 and snap-25 at 55 hpf. For all confocal images, developing motor neurons were examined from the same somites, as indicated. (B) Morphology of developing motor neurons under each of the indicated conditions. Arrows indicate increased branching after knockdown miR-153 ( miR-153 MO ) or overexpression snap-25a,b mRNA. Arrowheads indicate the structural defects after miR-153 overexpression or knockdown of snap-25a,b ( snap-25a,b MO ). Scale bar: 20 µm. (C) Quantification of motor neuron axonal branch number under the different conditions shown in (B). Error bars show s.e.m. Significance was determined using ANOVA with Dunnett’s post-test, n = 5. *, p<0.01; **, p<0.005. (D) Quantification of motor neuron axon length relative to uninjected control under the different conditions shown in (B). Error bars show s.e.m. ANOVA with Dunnett’s post-test, n = 5. *, p<0.05; **, p<0.01.

    Techniques Used: Transgenic Assay, Over Expression

    rabbit polyclonal anti snap 25  (Alomone Labs)


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    Alomone Labs rabbit polyclonal anti snap 25
    SYT-1 protein levels are unchanged, but <t>SNAP-25</t> protein expression was decreased in the cortex of NL3 R451C mice. SYT-1 and SNAP-25 protein expression in cortical ( A , G ), striatal ( C , I ) and cerebellar ( E , K ) lysates from NL3 R451C and WT mice were analysed via Western blot. Densitometric analysis was performed to demonstrate quantitative expression of SYT-1 ( B ,D, F ) and SNAP-25 ( H , J , L ) relative to β actin expression. Genotype differences were analysed using an unpaired, two-tailed Student’s t-test (n = 6 mice in each group); **p < 0.01. Data represented as mean ± SEM.
    Rabbit Polyclonal Anti Snap 25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti snap 25/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti snap 25 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "An altered glial phenotype in the NL3 R451C mouse model of autism"

    Article Title: An altered glial phenotype in the NL3 R451C mouse model of autism

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-71171-y

    SYT-1 protein levels are unchanged, but SNAP-25 protein expression was decreased in the cortex of NL3 R451C mice. SYT-1 and SNAP-25 protein expression in cortical ( A , G ), striatal ( C , I ) and cerebellar ( E , K ) lysates from NL3 R451C and WT mice were analysed via Western blot. Densitometric analysis was performed to demonstrate quantitative expression of SYT-1 ( B ,D, F ) and SNAP-25 ( H , J , L ) relative to β actin expression. Genotype differences were analysed using an unpaired, two-tailed Student’s t-test (n = 6 mice in each group); **p < 0.01. Data represented as mean ± SEM.
    Figure Legend Snippet: SYT-1 protein levels are unchanged, but SNAP-25 protein expression was decreased in the cortex of NL3 R451C mice. SYT-1 and SNAP-25 protein expression in cortical ( A , G ), striatal ( C , I ) and cerebellar ( E , K ) lysates from NL3 R451C and WT mice were analysed via Western blot. Densitometric analysis was performed to demonstrate quantitative expression of SYT-1 ( B ,D, F ) and SNAP-25 ( H , J , L ) relative to β actin expression. Genotype differences were analysed using an unpaired, two-tailed Student’s t-test (n = 6 mice in each group); **p < 0.01. Data represented as mean ± SEM.

    Techniques Used: Expressing, Western Blot, Two Tailed Test

    Antibodies used in Western blot analysis.
    Figure Legend Snippet: Antibodies used in Western blot analysis.

    Techniques Used: Western Blot

    rabbit polyclonal anti snap25  (Alomone Labs)


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    Alomone Labs rabbit polyclonal anti snap25
    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.
    Rabbit Polyclonal Anti Snap25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses"

    Article Title: A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2020.107968

    Increased Immunolabeling Efficiency in Epoxy-Resin-Embedded Tissue after Etching and Antigen Retrieval (A and B) Post-embedding fluorescent labeling for SNAP25 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.
    Figure Legend Snippet: Increased Immunolabeling Efficiency in Epoxy-Resin-Embedded Tissue after Etching and Antigen Retrieval (A and B) Post-embedding fluorescent labeling for SNAP25 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.

    Techniques Used: Immunolabeling, Labeling, Immunofluorescence, Fluorescence


    Figure Legend Snippet:

    Techniques Used: Plasmid Preparation, Recombinant, Software, Microscopy

    rabbit polyclonal anti snap 25  (Alomone Labs)


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    Alomone Labs rabbit polyclonal anti snap 25
    Rabbit Polyclonal Anti Snap 25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit polyclonal anti snap 25  (Alomone Labs)


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    Alomone Labs rabbit polyclonal anti snap 25
    Rabbit Polyclonal Anti Snap 25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    snap25  (Alomone Labs)


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    Alomone Labs snap25
    Syn-3 (A) and Syn-1A (C) interactions with the indicated Ca v α1 subunits (Ca v 1.2, Ca v 1.3, Ca v 2.3 and Ca v 2.2) and auxiliary subunits (α 2 δ-1 and β3) and <t>SNAP25</t> in INS-1 cells. Densitometric analysis of Syn-3 co-IP (B) and Syn-1A co-IP (D), expressed as percent recovery of total lysate inputs (which showed equal protein loading in ), shows that high glucose (16.7 mM) plus GLP-1 (10 nM) increased the association of these syntaxins with the respective Ca v s. Values are means±SEM, n = 3. *p<0.05, ***p<0.001, NS: not significant. As control (E) shows representative blots from five separate co-IP experiments with pre-immune IgG, which did not bring down syntaxins or Ca v s ( left lanes). Righ t lanes show the input lysates. All five experiments probed for the Ca v α subunits and α 2 δ-1, whereas β3, Syn-3 and Syn-1A were probed on two blots from separate experiments.
    Snap25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Syntaxin-3 Binds and Regulates Both R- and L-Type Calcium Channels in Insulin-Secreting INS-1 832/13 Cells"

    Article Title: Syntaxin-3 Binds and Regulates Both R- and L-Type Calcium Channels in Insulin-Secreting INS-1 832/13 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0147862

    Syn-3 (A) and Syn-1A (C) interactions with the indicated Ca v α1 subunits (Ca v 1.2, Ca v 1.3, Ca v 2.3 and Ca v 2.2) and auxiliary subunits (α 2 δ-1 and β3) and SNAP25 in INS-1 cells. Densitometric analysis of Syn-3 co-IP (B) and Syn-1A co-IP (D), expressed as percent recovery of total lysate inputs (which showed equal protein loading in ), shows that high glucose (16.7 mM) plus GLP-1 (10 nM) increased the association of these syntaxins with the respective Ca v s. Values are means±SEM, n = 3. *p<0.05, ***p<0.001, NS: not significant. As control (E) shows representative blots from five separate co-IP experiments with pre-immune IgG, which did not bring down syntaxins or Ca v s ( left lanes). Righ t lanes show the input lysates. All five experiments probed for the Ca v α subunits and α 2 δ-1, whereas β3, Syn-3 and Syn-1A were probed on two blots from separate experiments.
    Figure Legend Snippet: Syn-3 (A) and Syn-1A (C) interactions with the indicated Ca v α1 subunits (Ca v 1.2, Ca v 1.3, Ca v 2.3 and Ca v 2.2) and auxiliary subunits (α 2 δ-1 and β3) and SNAP25 in INS-1 cells. Densitometric analysis of Syn-3 co-IP (B) and Syn-1A co-IP (D), expressed as percent recovery of total lysate inputs (which showed equal protein loading in ), shows that high glucose (16.7 mM) plus GLP-1 (10 nM) increased the association of these syntaxins with the respective Ca v s. Values are means±SEM, n = 3. *p<0.05, ***p<0.001, NS: not significant. As control (E) shows representative blots from five separate co-IP experiments with pre-immune IgG, which did not bring down syntaxins or Ca v s ( left lanes). Righ t lanes show the input lysates. All five experiments probed for the Ca v α subunits and α 2 δ-1, whereas β3, Syn-3 and Syn-1A were probed on two blots from separate experiments.

    Techniques Used: Co-Immunoprecipitation Assay

    rabbit anti snap25  (Alomone Labs)


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    Alomone Labs rabbit anti snap25
    Rabbit Anti Snap25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti snap25  (Alomone Labs)


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    Alomone Labs rabbit anti snap25
    Rabbit Anti Snap25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    snap 25  (Alomone Labs)


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    Alomone Labs snap 25
    Knockdown of <t>SNAP-25,</t> SNAP-25/SNAP-23, and synaptotagmin I by RNA interference in PC12 cells. A: immunoblots were used to assess the levels of SNAP-25, SNAP-23, and β-actin in wild-type PC12 cells, in SNAP-25 knockdown PC12 cells, and in SNAP-25/SNAP-23 double-knockdown PC12 cells. Note that the SNAP-23 knockdown was incomplete. Equal amounts of protein were loaded per lane. B: immunoblots were used to assess the levels of synaptotagmin I in wild-type PC12 cells and in synaptotagmin I knockdown PC12 cells.
    Snap 25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Interaction of anesthetics with neurotransmitter release machinery proteins"

    Article Title: Interaction of anesthetics with neurotransmitter release machinery proteins

    Journal: Journal of Neurophysiology

    doi: 10.1152/jn.00666.2012

    Knockdown of SNAP-25, SNAP-25/SNAP-23, and synaptotagmin I by RNA interference in PC12 cells. A: immunoblots were used to assess the levels of SNAP-25, SNAP-23, and β-actin in wild-type PC12 cells, in SNAP-25 knockdown PC12 cells, and in SNAP-25/SNAP-23 double-knockdown PC12 cells. Note that the SNAP-23 knockdown was incomplete. Equal amounts of protein were loaded per lane. B: immunoblots were used to assess the levels of synaptotagmin I in wild-type PC12 cells and in synaptotagmin I knockdown PC12 cells.
    Figure Legend Snippet: Knockdown of SNAP-25, SNAP-25/SNAP-23, and synaptotagmin I by RNA interference in PC12 cells. A: immunoblots were used to assess the levels of SNAP-25, SNAP-23, and β-actin in wild-type PC12 cells, in SNAP-25 knockdown PC12 cells, and in SNAP-25/SNAP-23 double-knockdown PC12 cells. Note that the SNAP-23 knockdown was incomplete. Equal amounts of protein were loaded per lane. B: immunoblots were used to assess the levels of synaptotagmin I in wild-type PC12 cells and in synaptotagmin I knockdown PC12 cells.

    Techniques Used: Western Blot

    Knockdown of SNAP 25, SNAP-23, and synaptotagmin I were confirmed with immunofluorescence measurements. Wild-type PC12 cells (left) or synaptotagmin I, SNAP25, or SNAP-23/SNAP-23 knockdown cells (right) were stained with corresponding primary antibodies and then Alexa Fluor 594 (or Alexa Fluor 488)-labeled secondary antibodies. Synaptotagmin expression was examined in wild-type PC12 cells (A) and synaptotagmin knockdown cells (B). SNAP-25 expression was examined in wild-type PC12 cells (C), SNAP-25 knockdown cells (D), and SNAP-23/SNAP-25 double knockdown cells (E). SNAP-23 expression was examined in wild-type cells (F) or SNAP-23/SNAP-25 double-knockdown cells (G). Microscope settings and gain were identical for all panels.
    Figure Legend Snippet: Knockdown of SNAP 25, SNAP-23, and synaptotagmin I were confirmed with immunofluorescence measurements. Wild-type PC12 cells (left) or synaptotagmin I, SNAP25, or SNAP-23/SNAP-23 knockdown cells (right) were stained with corresponding primary antibodies and then Alexa Fluor 594 (or Alexa Fluor 488)-labeled secondary antibodies. Synaptotagmin expression was examined in wild-type PC12 cells (A) and synaptotagmin knockdown cells (B). SNAP-25 expression was examined in wild-type PC12 cells (C), SNAP-25 knockdown cells (D), and SNAP-23/SNAP-25 double knockdown cells (E). SNAP-23 expression was examined in wild-type cells (F) or SNAP-23/SNAP-25 double-knockdown cells (G). Microscope settings and gain were identical for all panels.

    Techniques Used: Immunofluorescence, Staining, Labeling, Expressing, Microscopy

    Amperometric events recorded from knockdown cell lines were similar to those from wild-type cells under our stimulation conditions. A: representative traces showing 2.5 min of data from wild-type PC12 cells and the 3 knockdown cell lines used in these studies, as indicated. B: averaged half-times of amperometric events showing no clear difference between the different cell lines (W-T, wild-type PC12 cells; Syt-1, synaptotagmin I knockdown; SNAP-25, SNAP-25 knockdown; SNAP-25/23, SNAP-25/SNAP-23 double knockdown). Data from 25 different cells were used for each group shown. C: average number of molecules of neurotransmitter released per amperometric event for the 4 different cell lines. No clear difference was observed. n = 25 for each of the 4 groups.
    Figure Legend Snippet: Amperometric events recorded from knockdown cell lines were similar to those from wild-type cells under our stimulation conditions. A: representative traces showing 2.5 min of data from wild-type PC12 cells and the 3 knockdown cell lines used in these studies, as indicated. B: averaged half-times of amperometric events showing no clear difference between the different cell lines (W-T, wild-type PC12 cells; Syt-1, synaptotagmin I knockdown; SNAP-25, SNAP-25 knockdown; SNAP-25/23, SNAP-25/SNAP-23 double knockdown). Data from 25 different cells were used for each group shown. C: average number of molecules of neurotransmitter released per amperometric event for the 4 different cell lines. No clear difference was observed. n = 25 for each of the 4 groups.

    Techniques Used:

    Isoflurane, but not propofol or etomidate, maintains its inhibitory effect on neurotransmitter release on SNAP-25 knockdown PC-12 cells. A: averaged number of events in the absence (“Control”) and presence of isoflurane (1 mM). Isoflurane produced a significant reduction of ∼54% in the number of amperometric events (*P = 0.03, n = 21). B: averaged number of events in the absence (“Control”) and presence of propofol (5 μM). Propofol reduced the number of amperometric events by ∼27%, which was not significant (P = 0.09, n = 31). C: averaged number of events in the absence (“Control”) and presence of etomidate (40 μM). Etomidate produced an insignificant reduction of ∼8% on the number of amperometric events (P = 0.73, n = 25). Data were normalized to the mean of the control group. Note that the inhibitory effect of isoflurane on neurotransmitter release was largely intact while the inhibitory effect of etomidate was significantly reduced on SNAP-25 knockdown PC12 cells. The effects of propofol on neurotransmitter release were moderately reduced on SNAP-25 knockdown PC12 cells.
    Figure Legend Snippet: Isoflurane, but not propofol or etomidate, maintains its inhibitory effect on neurotransmitter release on SNAP-25 knockdown PC-12 cells. A: averaged number of events in the absence (“Control”) and presence of isoflurane (1 mM). Isoflurane produced a significant reduction of ∼54% in the number of amperometric events (*P = 0.03, n = 21). B: averaged number of events in the absence (“Control”) and presence of propofol (5 μM). Propofol reduced the number of amperometric events by ∼27%, which was not significant (P = 0.09, n = 31). C: averaged number of events in the absence (“Control”) and presence of etomidate (40 μM). Etomidate produced an insignificant reduction of ∼8% on the number of amperometric events (P = 0.73, n = 25). Data were normalized to the mean of the control group. Note that the inhibitory effect of isoflurane on neurotransmitter release was largely intact while the inhibitory effect of etomidate was significantly reduced on SNAP-25 knockdown PC12 cells. The effects of propofol on neurotransmitter release were moderately reduced on SNAP-25 knockdown PC12 cells.

    Techniques Used: Produced

    The inhibitory effect of isoflurane and etomidate, but not propofol, on neurotransmitter release is reduced on SNAP-25/SNAP-23 knockdown PC12 cells. A: averaged number of events in the absence (“Control”) and presence of isoflurane (1 mM). Isoflurane produced an insignificant reduction of ∼18% in the number of amperometric events (P = 0.35, n = 30). B: averaged number of events in the absence (“Control”) and presence of propofol (5 μM). Propofol reduced the number of amperometric events by ∼51%, which is significant (*P = 0.01, n = 30). C: averaged number of events in the absence (“Control”) and presence of etomidate (10 μM). Etomidate did not show any reduction of the number of amperometric events (P = 0.39, n = 18). Data were normalized to the mean of the control group. Note that the inhibitory effects of etomidate and isoflurane, but not propofol, on neurotransmitter release were significantly reduced on SNAP-25/SNAP-23 knockdown PC12 cells.
    Figure Legend Snippet: The inhibitory effect of isoflurane and etomidate, but not propofol, on neurotransmitter release is reduced on SNAP-25/SNAP-23 knockdown PC12 cells. A: averaged number of events in the absence (“Control”) and presence of isoflurane (1 mM). Isoflurane produced an insignificant reduction of ∼18% in the number of amperometric events (P = 0.35, n = 30). B: averaged number of events in the absence (“Control”) and presence of propofol (5 μM). Propofol reduced the number of amperometric events by ∼51%, which is significant (*P = 0.01, n = 30). C: averaged number of events in the absence (“Control”) and presence of etomidate (10 μM). Etomidate did not show any reduction of the number of amperometric events (P = 0.39, n = 18). Data were normalized to the mean of the control group. Note that the inhibitory effects of etomidate and isoflurane, but not propofol, on neurotransmitter release were significantly reduced on SNAP-25/SNAP-23 knockdown PC12 cells.

    Techniques Used: Produced

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    Alomone Labs snap 25
    Plasmid-based expression of <t>shRNA-SNAP-25</t> . A) Plasmid map of pG418-shRNA, a plasmid designed to express shRNAs from the mouse U6 promoter. B) The two 56-base deoxyoligonucleotides used in the construction of pG418-shRNA-SNAP-25 are shown as they would be paired after annealing. The labels above and below the oligonucleotides indicate the source or the function of the nucleotides in the indicated regions. C) The predicted stem-loop structure of shRNA-SNAP-25 expressed from pG418-shRNA-SNAP-25.
    Snap 25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Alomone Labs rabbit polyclonal anti snap 25
    SYT-1 protein levels are unchanged, but <t>SNAP-25</t> protein expression was decreased in the cortex of NL3 R451C mice. SYT-1 and SNAP-25 protein expression in cortical ( A , G ), striatal ( C , I ) and cerebellar ( E , K ) lysates from NL3 R451C and WT mice were analysed via Western blot. Densitometric analysis was performed to demonstrate quantitative expression of SYT-1 ( B ,D, F ) and SNAP-25 ( H , J , L ) relative to β actin expression. Genotype differences were analysed using an unpaired, two-tailed Student’s t-test (n = 6 mice in each group); **p < 0.01. Data represented as mean ± SEM.
    Rabbit Polyclonal Anti Snap 25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs rabbit polyclonal anti snap25
    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.
    Rabbit Polyclonal Anti Snap25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs snap25
    Syn-3 (A) and Syn-1A (C) interactions with the indicated Ca v α1 subunits (Ca v 1.2, Ca v 1.3, Ca v 2.3 and Ca v 2.2) and auxiliary subunits (α 2 δ-1 and β3) and <t>SNAP25</t> in INS-1 cells. Densitometric analysis of Syn-3 co-IP (B) and Syn-1A co-IP (D), expressed as percent recovery of total lysate inputs (which showed equal protein loading in ), shows that high glucose (16.7 mM) plus GLP-1 (10 nM) increased the association of these syntaxins with the respective Ca v s. Values are means±SEM, n = 3. *p<0.05, ***p<0.001, NS: not significant. As control (E) shows representative blots from five separate co-IP experiments with pre-immune IgG, which did not bring down syntaxins or Ca v s ( left lanes). Righ t lanes show the input lysates. All five experiments probed for the Ca v α subunits and α 2 δ-1, whereas β3, Syn-3 and Syn-1A were probed on two blots from separate experiments.
    Snap25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs rabbit anti snap25
    Syn-3 (A) and Syn-1A (C) interactions with the indicated Ca v α1 subunits (Ca v 1.2, Ca v 1.3, Ca v 2.3 and Ca v 2.2) and auxiliary subunits (α 2 δ-1 and β3) and <t>SNAP25</t> in INS-1 cells. Densitometric analysis of Syn-3 co-IP (B) and Syn-1A co-IP (D), expressed as percent recovery of total lysate inputs (which showed equal protein loading in ), shows that high glucose (16.7 mM) plus GLP-1 (10 nM) increased the association of these syntaxins with the respective Ca v s. Values are means±SEM, n = 3. *p<0.05, ***p<0.001, NS: not significant. As control (E) shows representative blots from five separate co-IP experiments with pre-immune IgG, which did not bring down syntaxins or Ca v s ( left lanes). Righ t lanes show the input lysates. All five experiments probed for the Ca v α subunits and α 2 δ-1, whereas β3, Syn-3 and Syn-1A were probed on two blots from separate experiments.
    Rabbit Anti Snap25, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti snap25/product/Alomone Labs
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    Image Search Results


    Plasmid-based expression of shRNA-SNAP-25 . A) Plasmid map of pG418-shRNA, a plasmid designed to express shRNAs from the mouse U6 promoter. B) The two 56-base deoxyoligonucleotides used in the construction of pG418-shRNA-SNAP-25 are shown as they would be paired after annealing. The labels above and below the oligonucleotides indicate the source or the function of the nucleotides in the indicated regions. C) The predicted stem-loop structure of shRNA-SNAP-25 expressed from pG418-shRNA-SNAP-25.

    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: Plasmid-based expression of shRNA-SNAP-25 . A) Plasmid map of pG418-shRNA, a plasmid designed to express shRNAs from the mouse U6 promoter. B) The two 56-base deoxyoligonucleotides used in the construction of pG418-shRNA-SNAP-25 are shown as they would be paired after annealing. The labels above and below the oligonucleotides indicate the source or the function of the nucleotides in the indicated regions. C) The predicted stem-loop structure of shRNA-SNAP-25 expressed from pG418-shRNA-SNAP-25.

    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: Plasmid Preparation, Expressing, shRNA

    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.

    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

    Catecholamine secretion is reduced in SNAP-25 knockdown PC12 cells . Representative amperometric traces are shown from a control cell stably transfected with pG418-shRNA lacking an shRNA insert (empty vector) (A) and from a SNAP-25 knockdown cell prior to and during a 2.5 min stimulation with 60 mM KCl (B). To the right of each of the traces is an averaged amperometric event shown on an expanded time scale. C) When compared to empty vector controls (n = 18) the silencing of SNAP-25 in PC12 cells (n = 18) did not alter the number of norepinephrine molecules per release event. D) Silencing of SNAP-25 resulted in a 63% reduction in the total number of exocytotic events produced following stimulation. Control transfected cells and SNAP-25 knockdown cells produced a mean ± SEM of 79 ± 17 events and 29 ± 10 events, respectively. *p < 0.03 (Student's t-test). E) Transient transfection with a human SNAP-25 expression plasmid rescued the deficit in catecholamine secretion in the SNAP-25 knockdown cells. *p < 0.03 (Student's t-test).

    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: Catecholamine secretion is reduced in SNAP-25 knockdown PC12 cells . Representative amperometric traces are shown from a control cell stably transfected with pG418-shRNA lacking an shRNA insert (empty vector) (A) and from a SNAP-25 knockdown cell prior to and during a 2.5 min stimulation with 60 mM KCl (B). To the right of each of the traces is an averaged amperometric event shown on an expanded time scale. C) When compared to empty vector controls (n = 18) the silencing of SNAP-25 in PC12 cells (n = 18) did not alter the number of norepinephrine molecules per release event. D) Silencing of SNAP-25 resulted in a 63% reduction in the total number of exocytotic events produced following stimulation. Control transfected cells and SNAP-25 knockdown cells produced a mean ± SEM of 79 ± 17 events and 29 ± 10 events, respectively. *p < 0.03 (Student's t-test). E) Transient transfection with a human SNAP-25 expression plasmid rescued the deficit in catecholamine secretion in the SNAP-25 knockdown cells. *p < 0.03 (Student's t-test).

    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: Stable Transfection, Transfection, shRNA, Plasmid Preparation, Produced, Expressing

    SYT-1 protein levels are unchanged, but SNAP-25 protein expression was decreased in the cortex of NL3 R451C mice. SYT-1 and SNAP-25 protein expression in cortical ( A , G ), striatal ( C , I ) and cerebellar ( E , K ) lysates from NL3 R451C and WT mice were analysed via Western blot. Densitometric analysis was performed to demonstrate quantitative expression of SYT-1 ( B ,D, F ) and SNAP-25 ( H , J , L ) relative to β actin expression. Genotype differences were analysed using an unpaired, two-tailed Student’s t-test (n = 6 mice in each group); **p < 0.01. Data represented as mean ± SEM.

    Journal: Scientific Reports

    Article Title: An altered glial phenotype in the NL3 R451C mouse model of autism

    doi: 10.1038/s41598-020-71171-y

    Figure Lengend Snippet: SYT-1 protein levels are unchanged, but SNAP-25 protein expression was decreased in the cortex of NL3 R451C mice. SYT-1 and SNAP-25 protein expression in cortical ( A , G ), striatal ( C , I ) and cerebellar ( E , K ) lysates from NL3 R451C and WT mice were analysed via Western blot. Densitometric analysis was performed to demonstrate quantitative expression of SYT-1 ( B ,D, F ) and SNAP-25 ( H , J , L ) relative to β actin expression. Genotype differences were analysed using an unpaired, two-tailed Student’s t-test (n = 6 mice in each group); **p < 0.01. Data represented as mean ± SEM.

    Article Snippet: Rabbit polyclonal anti-SNAP-25 (23 kDa) , Alomone Labs #ANR-001 , 1:500.

    Techniques: Expressing, Western Blot, Two Tailed Test

    Antibodies used in Western blot analysis.

    Journal: Scientific Reports

    Article Title: An altered glial phenotype in the NL3 R451C mouse model of autism

    doi: 10.1038/s41598-020-71171-y

    Figure Lengend Snippet: Antibodies used in Western blot analysis.

    Article Snippet: Rabbit polyclonal anti-SNAP-25 (23 kDa) , Alomone Labs #ANR-001 , 1:500.

    Techniques: Western Blot

    Increased Immunolabeling Efficiency in Epoxy-Resin-Embedded Tissue after Etching and Antigen Retrieval (A and B) Post-embedding fluorescent labeling for SNAP25 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.

    Journal: Cell Reports

    Article Title: A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses

    doi: 10.1016/j.celrep.2020.107968

    Figure Lengend Snippet: Increased Immunolabeling Efficiency in Epoxy-Resin-Embedded Tissue after Etching and Antigen Retrieval (A and B) Post-embedding fluorescent labeling for SNAP25 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.

    Article Snippet: Rabbit polyclonal anti-SNAP25 , Alomone , Cat#ANR-001; RRID: AB_2040196.

    Techniques: Immunolabeling, Labeling, Immunofluorescence, Fluorescence

    Journal: Cell Reports

    Article Title: A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses

    doi: 10.1016/j.celrep.2020.107968

    Figure Lengend Snippet:

    Article Snippet: Rabbit polyclonal anti-SNAP25 , Alomone , Cat#ANR-001; RRID: AB_2040196.

    Techniques: Plasmid Preparation, Recombinant, Software, Microscopy

    Syn-3 (A) and Syn-1A (C) interactions with the indicated Ca v α1 subunits (Ca v 1.2, Ca v 1.3, Ca v 2.3 and Ca v 2.2) and auxiliary subunits (α 2 δ-1 and β3) and SNAP25 in INS-1 cells. Densitometric analysis of Syn-3 co-IP (B) and Syn-1A co-IP (D), expressed as percent recovery of total lysate inputs (which showed equal protein loading in ), shows that high glucose (16.7 mM) plus GLP-1 (10 nM) increased the association of these syntaxins with the respective Ca v s. Values are means±SEM, n = 3. *p<0.05, ***p<0.001, NS: not significant. As control (E) shows representative blots from five separate co-IP experiments with pre-immune IgG, which did not bring down syntaxins or Ca v s ( left lanes). Righ t lanes show the input lysates. All five experiments probed for the Ca v α subunits and α 2 δ-1, whereas β3, Syn-3 and Syn-1A were probed on two blots from separate experiments.

    Journal: PLoS ONE

    Article Title: Syntaxin-3 Binds and Regulates Both R- and L-Type Calcium Channels in Insulin-Secreting INS-1 832/13 Cells

    doi: 10.1371/journal.pone.0147862

    Figure Lengend Snippet: Syn-3 (A) and Syn-1A (C) interactions with the indicated Ca v α1 subunits (Ca v 1.2, Ca v 1.3, Ca v 2.3 and Ca v 2.2) and auxiliary subunits (α 2 δ-1 and β3) and SNAP25 in INS-1 cells. Densitometric analysis of Syn-3 co-IP (B) and Syn-1A co-IP (D), expressed as percent recovery of total lysate inputs (which showed equal protein loading in ), shows that high glucose (16.7 mM) plus GLP-1 (10 nM) increased the association of these syntaxins with the respective Ca v s. Values are means±SEM, n = 3. *p<0.05, ***p<0.001, NS: not significant. As control (E) shows representative blots from five separate co-IP experiments with pre-immune IgG, which did not bring down syntaxins or Ca v s ( left lanes). Righ t lanes show the input lysates. All five experiments probed for the Ca v α subunits and α 2 δ-1, whereas β3, Syn-3 and Syn-1A were probed on two blots from separate experiments.

    Article Snippet: All Ca v subunits antibodies are from Alomone Labs (Jerusalem, Israel), and SNAP25, Syn-1A and Syn-3 are from SySy (Goettingen, Germany); the specificity of these antibodies was well characterized by these companies, which been used broadly.

    Techniques: Co-Immunoprecipitation Assay