anr-10-idex-003-02  (IDEX)

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

Journal: CNS Neuroscience & Therapeutics

Article Title: Alpha‐lipoic acid downregulates TRPV1 receptor via NF‐κB and attenuates neuropathic pain in rats with diabetes

doi: 10.1111/cns.13303

Figure Lengend Snippet: ALA inhibited TRPV1 expression via suppressing NF‐κB. A, Western blots for NF‐κB in DRGs innervating the hindpaw from control and STZ‐induced diabetic rats. Bar graph involved mean density relative of β‐actin and NF‐κB from control and STZ‐induced diabetic rats. STZ injection significantly enhanced expression of NF‐κB (N = 4 for each group; * P < .05, compared with CNT, two‐sample t test) in L4‐L6 DRGs. B, Western blots for TRPV1 in L4‐L6 DRGs from diabetic rats treated with p65 siRNA lentivirus and NC siRNA lentivirus, respectively. Graph showed mean density relative to GAPDH for TRPV1. The lentiviruses were intrathecally injected into rats. The expression of TRPV1 significantly reduced after p65 siRNA lentiviruses treatment compared with NC siRNA group (N = 4 for each group, * P < .05, compared with NC siR, two‐sample t test). C, Western blots for NF‐κB in L4‐L6 DRGs from diabetic rats treated with NS and ALA, respectively. Graph showed mean density relative to GAPDH for NF‐κB. ALA treatment strongly reduced the expression of NF‐κB (N = 4 for NS group, N = 3 for ALA group, ** P < .01, compared with NS, two‐sample t test)

Article Snippet: Anti‐TRPV1 (#ACC‐030, Alomone, 1:200), anti‐p65 (#6956, Cell Signaling Technology, 1:1000), and corresponding horseradish peroxidase‐conjugated secondary anti‐rabbit and anti‐mouse antibodies at dilutions of 1:2000 and 1:2000 were used to probe the proteins, respectively.

Techniques: Expressing, Western Blot, Injection