Inc burlingame ca developed with cy3 conjugated

This preview shows 4 out of 6 pages.

Inc. Burlingame, CA), developed with Cy3- conjugated streptavidin (Jackson ImmunoResearch Lab. Inc. West Grove, PA) were used, and visual- ized by fluorescence microscopy with rhodamine filter set. Adenovirus Vector Construct The cDNA for human SMN (a gift from A. Burghes) (Monani et al. , 2000b) was subcloned into pIRES2-EGFP expression plasmid (Clontech, Mountain View, CA) containing a human cyto- megalovirus (CMV) early promoter, enhancer and internal ribosome entry signal (IRES), fol- lowed by an EGFP cDNA. This expression cas- sette was then cloned into a pAdTrack shuttle vector (He et al. , 1998). This plasmid was then linearized with Pme I restriction enzyme and co- transformed into E. coli BJ5183 cells with the pAdEasy-1 adenoviral backbone plasmid. Kanamycin resistance-selected recombinants were confirmed using Hind III restriction endo- nuclease polymorphism. The linearized recom- binant plasmid was then transfected into HEK 293 cells. Adenovirus vector (AdV) production and titration was performed as described previ- ously (Acsadi et al. , 1994). To verify AdV- derived SMN expression, NSC-34 cells were infected at 100 multiplicity of infection (M.O.I.). EGFP expression was monitored by epifluores- cent inverted microscopy (Nikon). Smn protein expression was confirmed using immunohis- tochemistry, and polyclonal anti-smn antibody (Transduction Labs) used to visualize intracel- lular smn expression.
Image of page 4

Subscribe to view the full document.

ANTI-APOPTOTIC EFFECT OF SMN ON NEURAL CELLS 43 Statistical Analysis For the cell viability assay, four replicated wells were used for each group. Experiments were repeat- ed in 3 plates under the same conditions. Each sur- vival measurement (MTT test; mean ± SD) was normalized to control group. For determining apop- tosis by TUNEL staining, at least three different fields were counted, and this was repeated three times independently. For real-time PCR, each tested sample was in triplicate and all experiments were done in triplicate. Western blotting experiments were also done in triplicate. The statistical signifi- cance was evaluated using Statistica (StatSoft Inc., 1998, Tulsa OK) or Excel (Microsoft). Variance for each percentage is reported as the ratio of the abso- lute mean value. N for each analysis is 3 unless indicated. Each study was repeated at least once. RESULTS siRNA Suppression of SMN Expression Quantitative real time RT-PCR was used to assay the ability of siRNA to suppress SMN expression (FIG. 1A). SMN transcripts isolated from NSC-34 cells were decreased by 32% relative to control siRNA 48 h after transfection ( p <0.01). The decrease was 63% of NSC-34 cell SMN expression 72 h after exposure to SMN siRNA compared to FIGURE 1 SMN mRNA and protein quantification after SMN stealth SiRNA (Invitrogen) treatment of NSC-34 cells ( N =4: both ** p <0.01 compared to respective controls). A: Real time PCR quantification of SMN gene expression.
Image of page 5
Image of page 6
You've reached the end of this preview.
  • Spring '10
  • Bier
  • Apoptosis, Cell culture, SMN

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern