The complete cDNA coding sequence of the sspaqr1 gene was obtained using reverse transcriptase polymerase chain reaction (RTPCR). For RTPCR, RNA was extracted as described previously [54]. The cDNA was obtained using the RETROscript™ First Strand Synthesis kit (Ambion, Applied Biosystems, Foster City, CA, USA) and used as template. : VLCLAYD(fw)/GGCDWYL(rev) primer pair. The sequence of these primers were the following: Repotrectinib mouse 5′ tatttgtgtctttcttac 3′ and 5′ ataccattaacaacagcc 3′, respectively.

The following PCR parameters were used: an initial denaturation step at 94°C for 30 sec, followed by 25 cycles of denaturation at 94°C for 5 sec, annealing at 40°C for 10 sec, and extension at 72°C for 2 min. The RTPCR products were cloned as described previously [54] and the inserts sequenced using commercial sequencing services

from Davis Sequencing (Davis, CA, USA). Bioinformatics sequence analysis The theoretical molecular weight of SsPAQR1 was calculated using the on-line ExPASy tool (http://expasy.org/tools/pi_tool.html). The protein classification was performed using the PANTHER Gene and Protein Classification System (http://www.PANTHERdb.org) [31]. On-line database search was performed with 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 [57]. Transmembrane domains were identified using TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM) [32] and visualized with TOPO2 (http://www.sacs.ucsf.edu/TOPO2/). SOSUI server (http://bp.nuap.nagoya-u.ac.jp/sosui/sosuiframe0E.html) and PSIPRED Protein Prediction server, MEMSAT-SVM

(http://bioinf.cs.ucl.ac.uk/psipred/) were also used to identify transmembrane domains [33, 34, 58]. Cellular localization of the SsPAQR1 was done using PSORT II Server (http://PSORT.ims.u-tokyo.ac.jp/) 3-oxoacyl-(acyl-carrier-protein) reductase [35] and for the identification of mitochondrial signal sequence Predotar (http://urgi.versailles.inra.fr/predotar/predotar.html) [36], TargetP 1.1 server (http://www.cbs.dtu.dk/services/TargetP) [37] and MitoProt (http://ihg.gsf.de/ihg/mitoprot.html) [59] servers were used. Multiple sequence alignments were built using MCOFFEE (http://igs-server-cnrs-mrs.fr/tcoffee/tcoffee_ cgi/index.cgi) [60]. The alignment in Additional file 1 was visualized using GeneDoc (http://www.psc.edu/ biomed/genedoc). The accession numbers of the sequences used for the multiple sequence alignment of G protein subunits were: S. ATM Kinase Inhibitor schenckii, ACA43006.1; M. oryzae, XP_362234.1; Trichoderma reesei, EGR51560.1; N. crassa, XP_965338.1; Chaetomium globosum, XP_001221101.1; F. oxysporum, EGU81989.