Experimental Procedures. During the past five (5) years Seller has not performed or permitted the performance of any Experimental Procedures involving patients in the Healthcare Facilities not authorized and conducted in accordance with the procedures of the applicable Institutional Review Board.
Experimental Procedures. No Seller has performed or authorized the performance of any experimental or research procedures or studies involving patients of any Facility that require the prior approval of any governmental entity that has not been obtained.
Experimental Procedures. Sellers have not performed or permitted the performance of any experimental or research procedures or studies involving patients of the Facilities not authorized and conducted in accordance with the procedures of the Institutional Review Board of the relevant Facility.
Experimental Procedures. The Seller Entities have not performed or permitted the performance of any experimental or research procedures or studies involving patients of any Hospital not authorized and conducted in accordance with the procedures of the Institutional Review Board of the relevant Hospital
Experimental Procedures. The study protocol was approved by the University of California, Berkeley Committee for Protection of Human Subjects, following the declaration of Helsinki. All partici- pants gave written informed consent after the nature of TABLE I. Individual and group lesion volume and percent damage to the left middle temporal gyrus (MTG) and superior temporal gyrus (STG), angular gyrus (AG) and supramarginal gyrus (SMG), and left inferior frontal gyrus (LIFG) Patient Lesion volume (cm3) % STG and MTG % AG and SMG % LIFG P1 18.32 26.9 7.4 0 P2 4.51 5.6 0 0 P3 93.75 62.3 48 0 P4 36.95 45.5 0 0 P5 103.17 22.7 21.7 21.4 P6 85.82 84.5 43.1 0 Mean (sd) 57.08 (38.68) 41.25 (28.82) 20 (21.4) 3.57 (8.74) Figure 2.
Experimental Procedures. The Seller Entities have not performed or permitted the performance of any experimental or research procedures or studies involving patients of the Hospital not authorized and conducted in accordance with the procedures of the Institutional Review Board of the Hospital. All human subject research (as that phrase is defined by federal Law), or research governed by the federal Animal Welfare Act, involving the Seller Entities is identified on Schedule 3.23.
Experimental Procedures. Expression constructs and antibodies Conventional molecular biological techniques (Sambrook et al. 1989) were used to subclone DNA fragments encoding torsinA WT, ΔE, Δ323-8, and NT into mammalian vectors expressing C-terminal HA, Myc, or FLAG tags for transfection into cells. A rabbit polyclonal anti-torsinA antibody was raised against the N-terminal region of torsinA and affinity purified as described previously (Chin et al. 2000; Xxxx et al. 2006). Other antibodies used in this study are as follows: anti-KDEL (Stressgen); anti-β-actin (Chemicon); mouse monoclonal anti-HA (12CA5); and anti-Myc (9E10) (Xxxx et al. 2006). Horseradish peroxidase-conjugated secondary antibodies were used for immunoblotting (Xxxxxxx Immunoresearch Laboratories, Inc.). Flourescein isothiocyanate (FITC)- and Texas Red (TR)-conjugated secondary antibodies were used for immunofluorescence microscopy (Xxxxxxx Immunoresearch Laboratories, Inc.). Cell transfections and co-immunoprecipitation Transfections of HeLa and SH-SY5Y cells with the indicated plasmids were performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Cell lysates were prepared from transfected cells and immunoprecipitations were carried out as described previously (Olzmann et al. 2004; Xxxx et al. 2006) using anti-HA or anti-Myc antibodies. Immunocomplexes were recovered by incubation with protein G-sepharose beads (Upstate). After washing, the immunocomplexes were analyzed by SDS-PAGE and immunoblotted with the appropriate antibodies and horseradish peroxidase-conjugated secondary antibodies. Results were visualized using enhanced chemiluminescence (ECL). Primary cell culture Primary cortical neuronal cultures were prepared from embryonic day 18 mice as described (Xxxxxx et al. 2004; Xxx et al. 2008) and maintained in NeuroBasal Media (Gibco) supplemented with AraC (Sigma) for 3-7 days. Mouse embryonic fibroblast cultures were prepared from embryonic day 13 mice by using a well-established method (Bi et al. 2004; Ma et al. 2004; Olzmann et al. 2007), and early passage cells were used for all experiments. Immunofluorescence confocal microscopy Cells were fixed in 4% paraformaldehyde, stained with appropriate primary and secondary antibodies and processed for indirect immunofluorescence microscopy as described previously (Olzmann et al. 2004; Olzmann et al. 2007; Xxx et al. 2008). Analysis and acquisition was performed using a Zeiss LSM 510 confocal laser-scanning microsc...
Experimental Procedures. Animals SR-BI-deficient mice were provided by Xx. X. Krieger (Massachusetts Institute of Technology, Boston, USA). Heterozygous SR-BI deficient (SR-BI +/-) mice were cross-bred to generate wild-type (SR-BI +/+) and homozygous SR-BI deficient (SR-BI -/-) progeny. The presence of the targeted and wild-type SR-BI alleles was assessed by PCR amplification of DNA extracted from tail biopsies (primers 5’-GAT-GGG-ACA-TGG-GAC-ACG-AAG-CCA- TTC-T-3’ and 5’-TCT-GTC-TCC-GTC-TCC-TTC-AGG-TCC-TGA-3’).The mice were fed a chow diet containing 4.3% (w/w) fat without added cholesterol (RM3, Hope Farms,Woerden, The Netherlands). Animal experiments were performed at the Gorlaeus Laboratories of the Leiden/Amsterdam Center for Drug Research in accordance with the national laws. All experimental protocols were approved by the Ethics Committee for Animal Experiments of the Leiden University. Erythrocyte parameters Blood (200µl) was collected in EDTA coated tubes (Sarstedt, Numbrecht, Germany) by tail bleeding of mice fasted overnight. Subsequently, the blood was analysed using a Sysmex XE- 2100 hematology analyzer (Sysmex Corp. Kobe, Japan) for erythrocyte counts, mean cellular volume (MCV), erythrocyte distribution width-standard deviation (RDW-SD), hematocrit, hemoglobin (HGB), mean cellular hemoglobin (MCH), and the mean cellular hemoglobin concentration (MCHC).
Experimental Procedures. The IRMC Entities have not performed or permitted the performance of any experimental or research procedures or studies involving patients not authorized and conducted in accordance with the procedures of the applicable Institutional Review Board.
Experimental Procedures. 2.2.1. Materials Materials were obtained from the following sources: Anti-FLAG M2 affinity gel and anti-FLAG M2 monoclonal antibody-peroxidase conjugate, bovine serum albumin (BSA), isoproterenol, U73122, L-(-)-Norepinephrine, penicillin, and streptomycin from Sigma Chemical Co. (St. Louis, MO); fetal bovine serum from Atlanta Biologicals (Atlanta, GA); trypsin, Dulbecco’s modified Eagle’s medium (DMEM) from Cellgro (Herndon, VA); Lipofectamine 2000 transfection reagent from Invitrogen (Carlsbad, CA); myo-[3H]inositol from American Radiolabeled Chemicals, Inc.(St. Louis, MO); RhoA G-XXXX™ Activation Assay colorimetric format kit and C3 exoenzyme from Cytoskeleton, Inc. (Denver, CO); xXXX XXXXX Kit (colorimetric) from Cell Biolabs, Inc. (San Diego, CA); conjugated goat anti-mouse monoclonal antibody from Rockland Inc. (Gilbertsville, PA); PTX was purchased from List Biologicals (Campbell, CA); p44/42 ERK1/2 (extracellular signal-regulated kinase 1/2) antibody, phospho-p44/42 ERK1/2 antibody, MEK1/2 inhibitor U0126, and bisindolymaleimide (BIS) from Cell Signaling Technology (Beverly, MA); Glu-Glu monoclonal antibody (anti-EE) from Covance, Inc. (Princeton, NJ), anti-Gαs, anti-Gαo anti-Gαi1, anti-Gαi2, anti-Gαi3 anti-Gα12, and anti- Gα13 antibodies from Santa Xxxx Biotechnology, Inc. (Santa Cruz, CA); anti-Gαq/11/14 antibody Z811 was kindly provided by Xx. Xxxx Xxxxxxxxx (U. Texas Southwestern, Dallas, TX); peroxidase-conjugated goat anti-mouse IgG antisera from Rockland Immunochemicals, Inc. (Gilbertsville, PA), and peroxidase-conjugated goat anti-rabbit was from Bio-Rad (Hercules, CA). The PAR-activating peptides (PAR-APs), XXXXX- XX0 (XXXXX) and 2-furoyl-LIGRLO-NH2 (LIGRLO), were synthesized by Xx. Xxx Xxxx at the Emory University Microchemical Facility (Atlanta, GA).
