Immunostaining of Spata33 protein in the juvenile (aged 10–20 days) and mature testes (aged 35 days) using anti-Spata33 antibody (A), and subcellular localization of Spata33 protein in the GC-1 cells and TM4 cells (B).

<p>A). Spata33 was expressed mainly in the spermatogonia (white arrow heads), spermatocytes (red arrows) and round spermatids (red arrow heads). The signals were observed in both the cytosol and nuclei from P12-P35, whereas no signals were detected in P10. In controls, no positive signals were observed in P35 testis sections when Spata33 antibody was replaced by 1% normal rabbit serum. Nuclei were re-dyed with Hematoxylin (blue). Bars, 20 µm. B). GC-1 and TM4 cells were transfected with Spata33-GFP (green, Excitation 488 nm, Emission 507 nm) and stained with Hoechst (blue, Excitation 352 nm, Emission 461 nm) respectively. Spata33 is localized both in the nuclei and cytoplasm within GC-1 cells or TM4 cells. Bar, 10 µm.</p

Expression pattern of <i>Spata33</i> in various tissues of adult mouse.

<p>A). RT-PCR of <i>Spata33</i> in adult tissues. <i>Hprt</i> was used as a standard. The gene <i>Spata33</i> was predominantly expressed in testis. Molecular weight is shown on the right. B). Real-time fluorescent quantitative PCR of the <i>Spata33</i> gene in adult tissues. Error bars indicate the standard deviation (SD) of the mean (n = 3). Y-axis represents relative expression levels of <i>Spata33</i> and X-axis shows different mouse tissues. C). Expression of Spata33 protein in various tissues. Mouse tissues were subjected to Western blot analysis with antibody against Spata33. Spata33 protein recognized a band at 15 KD. The Spata33 protein was predominantly expressed in testis. Actin was used as an internal control. Molecular weight is shown on the right. We repeated each experiment three times with independent individuals.</p

Phylogenetic tree, amino acid alignments, domains and modification sites of Spata33.

<p>A). Phylogenetic tree of Spata33 in mammals. Phylogenetic analysis was performed with Phylip. Numbers on the branches represent the bootstrap values from 1000 replicates obtained using the Neighbor-Joining method. The scale bar corresponds to the estimated evolutionary distance units. GenBank accession numbers are as follows: <i>Callithrix jacchus</i>, XP_002761326.1; <i>Canis lupus familiaris</i>, XP_003434741.1; <i>Cricetulus griseus</i>, XP_003495126.1; <i>Gorilla gorilla gorilla</i>, XP_004058209.1; <i>Homo sapiens,</i> BAG64150.1; <i>Macaca mulatta,</i> XP_001104069.2; <i>Mus musculus</i>, NP_796253.2; <i>Nomascus leucogenys</i>, XP_003280677.1; <i>Otolemur garnettii,</i> XP_003800884.1; <i>Pan paniscus</i>, XP_003805830.1; <i>Pan troglodytes</i>, XP_511172.4; <i>Papio anubis,</i> P_003917381.1; <i>Rattus norvegicus</i>, NP_001099665.1; <i>Saimiri boliviensis boliviensis</i>, XP_003944616.1; <i>Tupaia_chinensis</i>, ELW62868.1. B). Alignment of amino acid sequences of the Spata33 proteins. Amino acids that are identical in all these species are shown in white letters on black background. The DUF4609 domain predicted by Pfam is boxed. C). Schematic mapping of potential protein domains and post-translational modification sties. The predicted sites for N-myristoylation, N-glycosylation and phosphorylations in Spata33 were indicated (N-Myr, N-myristoylation site; N-Glyc, N-glycosylation site; S/T, Serine/Threonine phosphorylation sites).</p

Expression of <i>Spata33</i> mRNA and protein in the postnatal testes of mice.

<p>A). RT-PCR showed expression pattern of <i>Spata33</i> mRNA in testes from P2 to P20 (postnatal days). Total RNAs were isolated from mouse testes and then cDNAs were synthesized. <i>Hprt</i> was used as an internal control. Molecular weight is shown on the right. <i>Spata33</i> mRNA was increased markedly after P12. B). Real-time fluorescent quantitative PCR of the <i>Spata33</i> in testes from P2 to P20. Error bars indicate the standard deviation (SD) of the mean (n = 3). Y-axis represents relative expression levels of <i>Spata33</i> and X-axis shows different development stages P2-P20. C). Expression of Spata33 protein in testes from P2 to P20. Mouse testes were individually collected from aged 2 to 20 days and were subjected to Western blot analysis with antibody against Spata33. The protein level was markedly increased from P12. Actin was used as an internal control. Molecular weight is shown on the right. All experiments were performed three times with independent individuals.</p

The primers for sequencing.

<p>The primers for sequencing.</p

Primers for semi-quantitative RT-PCR analysis.

<p>Primers for semi-quantitative RT-PCR analysis.</p

MS results of the unknown proteins from mouse and the swamp eel.

<p>MS results of the unknown proteins from mouse and the swamp eel.</p

Sequence analysis of Hspa8b2 protein of the swamp eel.

<p><b>a.</b> Cartoon showed a linear representation of the swamp eel Hspa8b2 in comparison with Hspa8 of mouse and human. Different colors showed three conserved domains. Protein sequence in red square showed the corresponding sequence, which is similar to Hspa8 identified by MS analysis. <b>b.</b> A complete protein alignment of the swamp eel Hspa8b2 with Hspa8 of mouse and human using ClustalX V2.0 and Genedoc 2.7.0 (protein ID in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065269#pone.0065269.s002" target="_blank">Table S1</a>), and the positive and identity of the alignments were 100% and 86.7% respectively. White letters on black background indicated identical amino acids.</p

Phylogenetic tree of Hsp70 family in vertebrates.

<p>The phylogenetic tree was constructed using the Maximum Likelihood method based on the amino acids of Hsp70 members from human, mouse, rat, camel, cattle, platypus, chicken, frog, zebrafish, pufferfish, fugu, stickleback, medaka, tilapia and the swamp eel. The protein sequences were from NCBI and Ensembl database (protein ID in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065269#pone.0065269.s002" target="_blank">Table S1</a>). 14 proteins of the swamp eel had been grouped into 7 cluster of Hsp70 family (red arrows). The numbers at the nodes indicate bootstrap values.</p

Conserved domain analysis of Hsp70 family members in swamp eel.

<p>All the 14 proteins had NBD_sugar-kinase_HSP70_actin superfamily domain (green bars), and some genes had their own conserved domain (yellow bars). Hspa1a/b and Hspa8a1/a2/b1/b2 had a common domain (HSPA1–2_6–8-like_NBD). The numbers on the bars denote amino acid positions. GenBank accession numbers were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065269#pone.0065269.s002" target="_blank">Table S1</a>.</p