Journal: PLoS Neglected Tropical Diseases
Article Title: A Cytoplasmic New Catalytic Subunit of Calcineurin in Trypanosoma cruzi and Its Molecular and Functional Characterization
Figure Lengend Snippet: Genomic organization and transcription of T. cruzi CaNA2 gene. A) Southern blot hybridization. CL strain genomic DNA was digested with the indicated restriction enzymes, electrophoresed in agarose gel (1), transferred to Hybond-N membrane and hybridized to the 32 P-labeled TcCaNA2 gene probe (2). Size markers are indicated in kilobases (kb). B) Chromoblot hybridization. Chromosomal bands of epimastigotes from clone CL-Brener (CLB) and CL strain (CL) were separated by PFGE, stained with ethidium bromide (1), transferred to Hybond-N membrane and hybridized with the same probe described above (2). Size markers are indicated in megabase pair (Mbp). C) Northern blot hybridization. Total RNA (12 µg) extracted from different developmental forms of T. cruzi CL strain (TCT: tissue culture trypomastigotes, MT: metacyclic trypomastigotes, A: amastigotes, E: epimastigotes) was subjected to formaldehyde-agarose gel electrophoresis, stained with ethidium bromide (1), transferred to Hybond-N membrane and hybridized with the same probe described above (2). M, molecular size markers, are indicated in kilobases (kb). D) Amplification of TcCaNA2 gene by RT-PCR (upper) and immunoblotting of TcCaNA2 protein expression (lower) in different developmental forms of T. cruzi . M, molecular size markers (kb). (+): positive control, corresponds to plasmid pCR 2.1-TOPO with TcCaNA2.
Article Snippet: The amplification product was cloned in the vector pCR 2.1-TOPO (TOPO Cloning Vector Kit for sequencing, Invitrogen by Life Technologies) according to the manufacturer's instructions.
Techniques: Southern Blot, Hybridization, Agarose Gel Electrophoresis, Labeling, Staining, Northern Blot, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control, Plasmid Preparation, Polymerase Chain Reaction