Journal: The EMBO Journal

Article Title: An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer's disease

doi: 10.1038/sj.emboj.7600049

Figure Lengend Snippet: Selection of Rzs that suppress Tm-induced cell death from a randomized library. ( A ) Schematic representation of the selection system. ( B ) Number of transformants after each round of selection. Vectors isolated from surviving cells were introduced into DH5α. ( C ) Individual Rzs, selected from the randomized library, were examined for their ability to protect cells against Tm-mediated apoptosis. SK-N-SH cells that expressed selected Rzs were exposed to 2 μg/ml Tm for 24 h. Apoptosis was assessed by TUNEL staining. ( D ) The activation of caspase-3 depended on the activation of PKR. Cells were treated with Tm for 24 h or untreated (control), and then caspase-3 activity was measured. Cells treated with z-VAD-fmk were examined as controls.

Article Snippet: For the first screening, transfected cells were exposed to Tm for 24 h. For the second screening, cells were transfected with candidate plasmids from the first screening and collected after incubation with Tm for 48 h. Expression vectors harboring Rzs were isolated from surviving cells and used to transform DH5α (TOYOBO, Tokyo, Japan).

Techniques: Selection, Isolation, TUNEL Assay, Staining, Activation Assay, Activity Assay

Journal: Genes & Development

Article Title: The ROTUNDIFOLIA3 gene of Arabidopsis thaliana encodes a new member of the cytochrome P-450 family that is required for the regulated polar elongation of leaf cells

doi:

Figure Lengend Snippet: Physical map of the ROT3 locus and transcription of the wild-type gene. ( A ) Schematic map of the T-DNA-tagged rot3 gene. An arrow indicates the promoter sequence of the ROT3 gene. Recognition sites for restriction endonucleases Bam HI (B), Eco RI (E), and Hin dIII (H) are shown on the map. ( B ) Southern genomic DNA hybridization analysis of wild-type (wt) and rot3 alleles. Genomic DNA was digested with Eco RI and fragments after electrophoresis were allowed to hybridize with probe D shown in A. The hybridization pattern of rot3-1 indicates a deletion in the promoter and the first exon–intron region of the ROT3 locus. ( C ) RNA blot hybridization analysis of the wild-type plant 4 weeks after sowing. Hybridization of 1.5 μg of poly(A) + RNA with probe B shown in A. The result indicated that the transcribed RNA was 1.8 kb long. The membrane was exposed to an imaging plate for 1 week, and the band was visualized by an image analyzer (BAS1000; Fujix, Tokyo, Japan).

Article Snippet: For standard-stringency (high-stringency) Southern blot analysis, hybridi zation was carried out in 5× SSC, 0.5% SDS, 5× Denhardt’s solution, and 100 μg/ml denatured salmon sperm DNA at 65°C, with final washing in 0.1× SSC and 0.1% SDS at 65°C twice for 10 min. For reduced-stringency Southern blot analysis, hybridization was performed in the same solution as described above at 50°C, with final washing in 1× SSC and 0.1% SDS at 50°C twice for 15 min. For the plasmid rescue experiment, genomic DNA was isolated from the rot3-3 line, digested to completion with either Eco RI or Sal I, self-ligated by T4 DNA ligase (Toyobo, Tokyo, Japan), and used to transform E. coli DH5α cells as described previously ( ).

Techniques: Sequencing, DNA Hybridization, Electrophoresis, Hybridization, Northern blot, Imaging

Journal: Genes & Development

Article Title: The ROTUNDIFOLIA3 gene of Arabidopsis thaliana encodes a new member of the cytochrome P-450 family that is required for the regulated polar elongation of leaf cells

doi:

Figure Lengend Snippet: Southern genomic DNA hybridization analysis of the ROT3 gene. ( A,B ) Two blots with similar sets of restriction fragments of genomic DNA were allowed to hybridize with 32 P-labeled ROT3 cDNA with high-stringency conditions ( A ) and with low-stringency conditions ( B ) for hybridization and washing (see Materials and Methods for details). Note the presence of additional bands ( B, arrowhead) after hybridization and washing under low-stringency conditions.

Article Snippet: For standard-stringency (high-stringency) Southern blot analysis, hybridi zation was carried out in 5× SSC, 0.5% SDS, 5× Denhardt’s solution, and 100 μg/ml denatured salmon sperm DNA at 65°C, with final washing in 0.1× SSC and 0.1% SDS at 65°C twice for 10 min. For reduced-stringency Southern blot analysis, hybridization was performed in the same solution as described above at 50°C, with final washing in 1× SSC and 0.1% SDS at 50°C twice for 15 min. For the plasmid rescue experiment, genomic DNA was isolated from the rot3-3 line, digested to completion with either Eco RI or Sal I, self-ligated by T4 DNA ligase (Toyobo, Tokyo, Japan), and used to transform E. coli DH5α cells as described previously ( ).

Techniques: DNA Hybridization, Labeling, Hybridization