2704 Nucleic Acids Research, 2011, Vol. 39, No. 7 three runs. CT values were calculated using the average CT from each sample. Fold differences were calculated using the comparative CT method ( CT), using fold difference = 2 CT, as directed by the ABI 7300 qRT-PCR manual. Protein extract preparation, SDS–PAGE and western blotting Drosophila tissues were dissected in 0.7 PBS supple- mented with Mini Complete protease inhibitor cocktail without EDTA (Roche) and immediately frozen on dry ice. S2 cells were cultured as described above. Pelleted S2 cells were homogenized and lysed in RIPA buffer sup- plemented with Mini Complete protease inhibitor cocktail without EDTA (Roche) and microcentrifuged at maximum speed for 5 min. Soluble fractions were quantitated using the DC protein assay system (BioRad). Soluble extract (10–15 mg) or insoluble fractions were mixed with reducing SDS-sample buffer and proteins separated by SDS–PAGE (5% stacking gel; 12.5% separating gel) at 200 V and electro-transferred onto 0.2 mm Westran S PVDF membrane (Whatman) at 100 V for 1 h. After blocking with 5% non-fat dry milk (NFDM) in 1 PBS with 0.1% Tween-20 (5% NFDM) for 1 h, membranes were incubated overnight with primary Ab (1:1000 in 3% NFDM) at 4 C. HRP-conjugated second- ary Abs (1:50 000) were incubated at 4 C for 2 h. Chemilluminescent detection was achieved using ECL- Plus (GE Healthcare) and BioMax Chemilluminescence ﬁlm (Kodak). Northern analysis Embryonic, larval, and adult poly(A+) mRNAs (15 mg; Clontech) were resolved on a 1.5% formaldehyde agarose gel in MOPS buffer and blotted onto a 0.2 mm Optitran nitrocellulose membrane. Filters were hybridized with 32P-labelled oligonucleotide probes complementary to coding regions within rpL22-like (Table 1) according to hybridization conditions in Sambrook et al. (16). RNAs were visualized by phosphorimaging. Filters were stripped in boiling 0.1% SDS and re-hybridized as above. Size estimates for detected RNAs were determined using RNA markers (Promega or Invitrogen). Immunohistochemistry Testis squashes and IHC preparation was performed as previously reported (17,18) with minor modiﬁcations. Testes from mature adult wild-type ﬂies of mixed age (2–7-day old) were dissected in 0.7% saline, squashed and quickly frozen on dry ice. Tissues were ﬁxed and permeablized in ice-cold ethanol for 10 min and in 4% formaldehyde (in 1 PBS) for 7 min at room temperature (RT), followed by 2 15 min washes in PBS with 0.3% Triton X-100 and 0.3% sodium deoxycholate and a single wash of PBT (PBS with 0.1% Triton X-100). Blocking occurred for 1 h with PBTB (PBT with 3% BSA) at RT. Squashes were incubated overnight at 4 C with primary Ab (1:100 in PBTB) in a humid chamber and washed four times for 15 min in PBTB at RT. Alexa Fluor-conjugated secondary Abs (1:200 in PBTB) were incubated at 4 C in a humid chamber for 1 h and then washed four times for 10 min in PBTB at RT. DAPI staining (0.2 mg/ml in PBS) was performed after ﬁnal Ab washes for 5 min, washed twice for 1 min in PBS and mounted in Cytoseal 60 (Richard-Allan Scientiﬁc). Fluorescent micrographs were taken using Nikon Eclipse TE200U inverted ﬂuores- cence microscope coupled to a digital CCD camera. Polysome analysis Ribosome extracts were prepared using modiﬁed proced- ures from Qin et al. (19), Houmani and Ruf (20) and Pelczar and Filipowicz (21). Adult male Canton S ﬂies (2.37g) were homogenized in lysis buffer [1:5 w/v; (19)] and homogenates clariﬁed by centrifugation at 10K rpm for 10 min. Approximately 100 OD260 U/ml were loaded onto a 10–50% linear sucrose gradient [prepared by the horizontal method described in ref. (20)] and spun at 35K rpm for 160 min in a SW-41 rotor. Gradient fractions (0.5 ml) were collected and read at OD260 using a DU-800 spectrophotometer. Fractions were pooled and pelleted with equal volumes of 20 mM Tris–HCl, pH 7.2 and spun at 40K rpm for 12 h in an SW41 rotor. Pellets were resuspended in 20 mM Tris–HCl, pH 7.2 and sub- jected to SDS–PAGE and western analysis. RESULTS rpL22-like mRNA transcripts are enriched in the testis Gene expression proﬁling of embryonic germline stem cells (8) and adult ovary germline stem cells (10) previ- ously identiﬁed a novel and possible germline stem cell-speciﬁc gene known as rpL22-like (due to its similarity to rpL22; Figure 1A). An important goal was to reﬁne previously reported rpL22-like mRNA expression proﬁles (8–10) in order to initiate studies on RpL22-like protein expression and function. By RT-PCR analysis, we determined that rpL22-like is expressed not only in embryos, larvae and adults, but also in an embryonic- derived S2 cell line, as well as within gonads and heads of both sexes (Figure 2). The expected amplicon of 939 bp was present in all samples using primers determined by BLAST analysis to target only rpL22-like in the D. melanogaster genome. Interestingly, in addition to the expected RT-PCR product for rpL22-like there was a prominent lower MW amplicon of 392 bp that was reproducibly ampliﬁed in multiple experiments for all RNA samples analysed (Figure 2). Molecular character- ization (cloning and sequencing) of both amplicons indicated that both are derived from the rpL22-like gene. We refer to this lower MW amplicon as ‘rpL22-like short’ (described in detail below). Based on RT-PCR data, rpL22-like isoforms were detected in a variety of developmental stages and tissues. To determine isoform abundance in ﬂy gonads, heads and S2 cells, we used qRT-PCR and isoform-speciﬁc primers on different RNA samples (Figure 3; Supplementary Table S2), showing that both rpL22-like and rpL22-like short mRNAs are enriched in testis compared with other tissues examined. These data corroborate previously pub- lished microarray analyses (11) and high-throughput