2706 Nucleic Acids Research, 2011, Vol. 39, No. 7 lowest amounts in testes and in S2 cells, respectively. Steady-state levels of both rpL22-like mRNA isoforms are therefore highest in the testis. RpL22-like protein is differentially expressed and found in active ribosomes Based on the relative abundance of rpL22-like mRNA, we determined that RpL22-like protein expression would best Figure 2. RT-PCR analysis of rpL22-like in different developmental stages, tissues, and in S2 cells. RT-PCR using rpL22-like primers to amplify the coding sequence resulted in the expected 939 bp amplicon (rpL22-like, arrow) in all samples. An additional smaller amplicon (rpL22-like short, arrowhead) of 390 bp was present in all samples. Variability in the intensity of rpL22-like short was noted in numerous replicates of the RT-PCR for various samples as noted here for embryonic and adult samples. NTC: no template control; E: embryo; L: larva; A: adult; S2: S2 cells; T: testis; O: ovary; MH: male heads; FH: female heads; M: pGEM marker. be analysed in testes compared with other tissues. Since no RpL22-like Abs were previously available, paralogue- speciﬁc polyclonal Abs targeting C-terminal amino acid residues were designed for recognition of RpL22 or RpL22-like (Figure 1A). The C-terminal peptide Ab for RpL22 is identical to that used successfully by Ni et al. (14) in IHC experiments and ChIP analysis; therefore, we anticipated that this Ab would be useful in our protein blots and IHC studies to detect RpL22. The C-terminal Ab for RpL22-like recognition in protein blots and IHC studies was similarly based on the location of the Ab epitope for paralogue RpL22. We ﬁrst conﬁrmed that our Abs were speciﬁc for the proteins of interest by preimmune sera analysis and detec- tion of recombinant tagged proteins (Supplementray Figures S2 and S3). Western blot analysis was used to screen adult gonads, larval salivary glands, S2 cells and ﬂy heads for the presence of RpL22. As RpL22 is ubiqui- tously expressed, we expected that RpL22 would be detected in all tissues. Two prominent immunoreactive species, one at the predicted MW ( 33 kDa) for RpL22 and the other at 50 kDa (the latter seen in all tissues), were identiﬁed (Figure 4A). The amount of 50 kDa species varied in different tissues in multiple experiments and may represent incorporation of RpL22 into an SDS-resistant complex. Interestingly, relatively little, if any RpL22 of the expected size is found in the testis and in heads. Further, western analysis conﬁrmed that RpL22-like expression within the gonad is conﬁned to males, as a protein of the expected MW ( 34 kDa) is highly Figure 3. qRT-PCR reveals rpL22-like mRNA enrichment in testis. Using isoform-speciﬁc primers, qRT-PCR shows that both isoforms are more highly expressed in testis compared with other tissues. Vasa and 2-tubulin serve as germ cell- and testis-speciﬁc controls, respectively. Numbers in table represent CT values. *P < 0.01.