Figure 4. Western blot analysis conﬁrms differential expression of RpL22-like. (A) Compared to the recombinant standard (RS), western analysis for RpL22 shows immunodetection at either the expected MW of 32.9 kDa (arrow) and/or at a higher MW at 50 kDa in all tissues. Immunodetection of RpL22-like at the expected MW of 34.3 kDa (arrow- head) is solely visible in testes. Insoluble extracts from male and female heads contain a higher MW species of RpL22-like [whole intact heads of mixed sex with eyes (HI)]. No immunoreactive species is seen in eyeless heads (eyes are dissected out; EH) or in a soluble head extract. The additional lower band in the RS sample is endogenous bacterial protein recognized by mouse antisera (see Supplementary Figure S2 for additional explanation). (B) The electrophoretic shift in RpL22-like is not sex speciﬁc as seen by western analysis of whole head tissue from males (M) and females (F). S2: S2 cells; SG: larval salivary glands; T: testis; O: ovary; HS: soluble head extract. enriched in testis tissue and not ovary (Figure 4A). We also noted an immunoreactive product (found in lower abundance than in testes) in an insoluble head extract (only a very limited amount of this product is seen in a soluble head fraction in some preparations). Surprisingly in head tissue, immunodetection is seen at a higher MW ( 60 kDa). What factors contribute to the electrophoretic shift for both RpL22 and RpL22-like are unknown, but may include post-translational modiﬁcations or assembly into SDS-resistant complexes. In fact, in silico analysis for both RpL22 and RpL22-like predicts several sites for post-translational modiﬁcations, particularly for phos- phorylation [Eukaryotic Linear Motif resource for func- tional sites in proteins (ELM; http://elm.eu.org/)]. Unlike the protein expression pattern in the gonad, RpL22-like is detected in both male and female heads (Figure 4B). Detection of RpL22-like in soluble and insoluble frac- tions in different tissues, coupled with a difference in some aspect of structural conﬁguration (accounting for the higher MW) may indicate that RpL22-like has a different function in different tissues. Alternatively, the protein may have the same function in different tissues, but its subcellular distribution may be subject to speciﬁc regulation. Nucleic Acids Research, 2011, Vol. 39, No. 7 2707 The absence of RpL22-like detection in an extract from heads in which eyes were excised (Figure 4A) suggests that RpL22-like may be expressed in the eye; however, removal of eyes from ﬂy heads sometimes removes underlying brain medulla tissue as well. We were unable to resolve this issue by analysing extracts from isolated eyes due to eye pigment interference in protein fractionation and western blot analyses. Instead, we analysed the amount of RpL22-like in eyeless (ey) mutant heads where the amount of eye tissue is signiﬁcantly reduced compared to wild-type, and determined that there is a signiﬁcant decrease in the amount of RpL22-like in quantitatively similar amounts of head protein (Supplementary Figure S4). Resolution of RpL22-like expression in the head/eye awaits IHC analyses currently in progress. Given that RpL22-like is enriched in testis, we used IHC to conﬁrm its presence in the testis and to determine its localization relative to RpL22 by using the same paralogue-speciﬁc Abs as were used for protein blots. Immunohistochemistry on the adult male reproductive tract conﬁrmed that RpL22-like expression is conﬁned to testes and is not detected within seminal vesicles, acces- sory glands or the ejaculatory duct (Figure 5). RpL22-like is present within all stages of sperm cells, contained within testes [see extruded sperm (ES) and testis internal contents]. In contrast, RpL22 is expressed throughout the tract, including sperm cells. A more detailed descrip- tion of the staining patterns for RpL22 and RpL22-like within different cell types in testes and the reproductive tract is forthcoming in a separate report (M.G. Kearse and V.C. Ware, in preparation). Comparison of the amino acid sequence of RpL22-like with that of RpL22 shows conservation of documented functional residues [(20); Figure 1A] though their func- tionality within RpL22-like has not been conﬁrmed. Yet, we might predict that RpL22-like is an RNA binding RP competent for nucleolar import. In order to resolve this fundamental question about RpL22-like function, we per- formed western analysis on pooled fractions (from whole male ﬂies) containing polysomes and 80S ribosomes isolated on 10–50% sucrose density gradients (Figure 6). Both RpL22 and RpL22-like are detected in 80S ribo- some and polysome fractions, indicating that both proteins are stably associated with ribosomes. As RpL22-like is highly abundant in testis, we infer that the protein is a component of testis ribosomes. The presence of RpL22 in ribosome fractions from male extracts, however, does not verify that RpL22 functions as an RP in the testis; this determination will require additional analyses. Interestingly, only RpL22 of the expected MW is detected in ribosome and polysome fractions. The higher MW protein detected in Western blots is not an apparent component of ribosomes, suggesting that this RpL22-containing component may have a non-ribosomal role. rpL22-like short is an alternatively spliced mRNA variant Only one annotated transcript has previously been reported in Flybase for rpL22-like; therefore, the iden- tiﬁcation of additional amplicons was not anticipated.