For RTPCR the following primers were used: GPDH-UP-KMVV (fw) 5′ caaaatggttgtcaaggc 3′ and GAPDH-LW-ISPRI (rev) 5′ aaatccgtgggctgatcc 3′. Bioinformatics Sequence Analysis The theoretical molecular weights of the proteins were calculated using the on-line ExPASy tool (http://expasy.org/tools/pi_tool.html). On-line Prosite Scan (Proscan) (http://expasy.org/tools/scanprosite/), Pfam (http://pfam.sanger.ac.uk/search)
and Blocks (http://blocks.fhcrc.org/blocks/blocks_search.html) searches were used to identify potential motifs present in SsSOD, SsGAPDH, SsSit and SsNramp [41, 43, 78]. The protein classification was SAHA HDAC purchase performed using the PANTHER Gene and Protein Classification System (http://www.PANTHERdb.org) [38]. On-line database searches and comparisons for SsSOD, SsGAPDH, SsSit and SsNramp were performed with Integrated Protein Classification (iProClass) database (http://pir.georgetown.edu/pirwww/dbinfo/iproclass.shtml) [79] and the BLAST algorithm (http://www.ncbi.nlm.nih.gov/BLAST/) with a cutoff of 10-7, a low complexity filter and the BLOSUM 62 matrix [37]. Transmembrane helices were identified using the TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM) [80] and visualized with TOPO2 (http://www.sacs.ucsf.edu/TOPO2/). Cellular localization of the SsSOD and SsNramp was done
using the www.selleckchem.com/products/Temsirolimus.html PSORT II Server (http://PSORT.ims.u-tokyo.ac.jp/) [39] and the TargetP 1.1 server (http://www.cbs.dtu.dk/services/TargetP) [40]. Multiple sequence alignments were built using MCOFFEE (http://www.tcoffee.org)
[81, 82]. The alignments in Additional Files 1 and 3 to 5 were visualized using the program GeneDoc (http://www.psc.edu/biomed/genedoc). Vasopressin Receptor Acknowledgements The authors wish to acknowledge the technical support of Ms. Claribel González in the completion of the sssod gene sequence, Dr. Shirley ValentínSelleck Erastin -Berrios for the construction of the cDNA yeast two-hybrid library used to identify SOD and Dr. Mary H. Mays Serpan for editing this manuscript. This work is part of the Doctoral Dissertation requirement of LPS. This investigation was supported partially by the RISE Program grant R25GM061838 and by the National Institute of General Medicine, Minority Biomedical Research Support Grant 3S06-GM-008224. RGM acknowledges funding through NIH NIGMS grant T36GM008789-05 and acknowledges the use of the Pittsburgh Supercomputing Center National Resource for Biomedical Supercomputing resources funded through NIH NCRR grant 2 P41 RR06009-16A1. Electronic supplementary material Additional file 1: Protein multiple sequence alignment of SsSOD to other fungal SOD homologues. Multiple sequence alignment of the predicted amino acid sequence of S. schenckii SsSOD and SOD homologues from other fungi.