Journal:

Article Title: A Gene Coding for Tomato Fruit ?-Galactosidase II Is Expressed during Fruit Ripening

doi:

Figure Lengend Snippet: Detection of β-galactosidase activity from pTomβgal 4 expression in E. coli. Cells were harvested and extracts were prepared every 12 h and the A615 was measured. Cultures were grown with the addition of the chromogenic substrate X-Gal (open symbols) or X-Gal and the transcriptional inducer IPTG (closed symbols) in the medium. The vector used as a positive control for E. coli β-galactosidase activity was pGEM (▪), and the vector used as a negative control and for expression was pFLAG-CTC either without (○, •) or with (⋄, ♦) the pTomβgal 4 ORF.

Article Snippet: As a positive control for maximal β-galactosidase activity the vector pGEM (containing the E. coli lacZ gene fragment, Promega) was transformed into the strain DH5α (containing the lacI q Z ΔM15 cassette).

Techniques: Activity Assay, Expressing, Plasmid Preparation, Positive Control, Negative Control

Journal:

Article Title: Bcl-2 antiapoptotic protein mediates verotoxin II-induced cell death: possible association between Bcl-2 and tissue failure by E. coli O157:H7

doi:

Figure Lengend Snippet: VTII-Bcl-2 complex formation. (A) VTII, but not VTI, contains the pentameric sequence from the Bcl-2 BH1 domain. Shown are the schematic model, sequence alignment, and protein identities and similarities of human Bcl-2, VTI and VTII. The dotted line indicates the region of splicing variants. (B,C). VTII, but not VTI, interacts with Bcl-2. Expression of Bcl-2 in V (-) and B10 (+) cells was detected with immunoblotting (IB). B10 (B) and/or V (B,C) cell extracts were separated by SDS-PAGE, and far-western blotting (FW) was performed with biotinylated peptides (NWGRL and NWGRI in B) or VTs (VTI and VTII in C). Interacted protein was detected with avidin-HRP and ECL. Bars on the left show positions of protein markers 46, 30, 20, and 14 kDa, from top to bottom. (D) VTII interacts with Bcl-2 through the NWGRI sequence. Far-western blotting analysis was performed with biotinylated VTII and B10 cell extracts in the presence of NWGRI (0, 1, 5, and 10 nm). (E) Coimmunoprecipitation analysis. V (-) or B10 (+) cells were treated with VTII, and then immunoprecipitates with antibodies for Bcl-2 (Bcl-2 IPs) or VTII (VTII IPs) were separated on SDS-PAGE, and immunoblotting analysis with VTII antibody was performed (VTII WB).

Article Snippet: After washing with Tween-PBS, the membranes were incubated with a 1000-fold diluted biotinylated second antibody, washed with Tween-PBS, and then incubated with avidin-HRP (Vector Laboratories, Burlingame, CA) at room temperature for 1 hour.

Techniques: Sequencing, Expressing, Western Blot, SDS Page, Far Western Blot, Avidin-Biotin Assay