Gel shift assays Adult testis cytoplasmic extracts (30 mg) were incubated with [32P]-labeled RNA probes as previously described (9) and fractionated in 4% polyacrylamide gels in 0.5 Tris/ Borate/EDTA (TBE) buﬀer. For supershift assays, anti- bodies were preincubated with extracts for 5 min before addition of the radiolabeled RNA. Protein–protein interaction assays Recombinant PTBP2 (9) containing a His tag at its C-terminus was incubated with His-tag aﬃnity resins for 30 min at 48C in 200 ml of binding buﬀer containing 20 mM Tris–HCl, pH 7.6, 100 mM NaCl, 2 mM DTT and 0.5% Tween 20. Adult testis extract was added and incubated for an additional 30 min at 48C with rotation. The mixture was centrifuged for 1 min at 100g at 48C, and the pellet was washed three times with binding buﬀer. The pellets were boiled in sodium dodecyl sulfate (SDS) loading buﬀer for 3 min, and proteins were resolved in a 10% SDS–polyacry- lamide gel. KSRP was detected by western blotting. For the GST pull-down assay, the 52-kDa recombi- nant KSRP (t-KSRP) containing a GST tag was incuba- ted with recombinant PTBP2 for 30 min at 48C in 200 ml binding buﬀer. Glutathione agarose beads were added to the mixture and incubated for 30 min at 48C. The mixture was centrifuged and washed as described above. The pel- lets were boiled in SDS loading buﬀer and electrophoresed in a 10% SDS–polyacrylamide gel. PTBP2 was detected by western blotting. RNA immunoprecipitation assay Assays were performed as previously described (9,21). Testis cytoplasmic extracts (10 mg) from sexually mature CD-1 mice were precleared with protein A/G agarose beads (100 ml), preimmune serum (10 ml) and yeast tRNA (100 mg/ml) [prepared in the presence of SUPERase-in (1 U/ml), Ambion] and then incubated with 100 ml anti- KSRP [ab5, raised against the full-length KSRP (27)] and protein G agarose beads at 48C for 2 h. The mixtures were centrifuged at 400g for 5 min, and the pellets were washed and extracted in Trizol. Immunoprecipitated RNAs were reverse transcribed with random primers with the Superscript III reverse transcription kit (Invitrogen), and PCR assays were performed. Northern blot analysis For KSRP northern blots, total testicular RNAs (15 mg) were electrophoresed in 1% agarose–formaldehyde gels and transferred to Hybond-N + membranes (Amersham Biosciences) in alkaline buﬀer. The probe for KSRP and b-actin was labeled using Ready-to-go DNA labeling beads (Amersham Biosciences), and hybridizations were performed in Quikhyb buﬀer (Stratagene). Immunohistochemistry and western blot analyses Testes from sexually mature CD-1 mice were ﬁxed and processed by the Histological Core Facility of the Children’s Hospital of Pennsylvania. Immunohistochem- istry was performed as previously described (9). Nucleic Acids Research, 2008, Vol. 36, No. 22 7159 Anti-KSRP and anti-PTBP2 were diluted 1:200 before use. For western blotting, aliquots from tissue lysates or populations of isolated cells (20 mg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electro- phoresis (SDS–PAGE) and blotted onto polyvinylidene diﬂuoride membranes (Millipore, Bedford, MA). The indi- vidual cell types were isolated as previously reported (28). Anti-KSRP [DB-KS (11), raised against the C-terminal of KSRP, or a polyclonal antibody from Abnova (H00008570-A01)], at dilutions of 1:3000, or anti-b-actin (1:10,000 dilution) (Sigma, St Louis, MO) were used as primary antibodies. Immunodepletion and decay assays In vitro decay and transfection assays were performed as previously described (9). KSRP was removed from testis cytoplasm extracts by overnight immunoprecipitation at 48C with anti-KSRP [ab5, raised against the full-length KSRP (27)] that was bound to protein G-Sepharose. Equal amounts of supernatant from extracts, previously incubated with preimmune serum or anti-KSRP was used in the RNA decay assays. Transfection assays were performed as previously described (9). In brief, reporter vectors, pEGFP-C2- UTR or pEGFP-C2-F3 (9), were transfected into HeLa cells using Liposome 2000 Reagent (Invitrogen) following the manufacturer’s protocol. Where noted, reporter vec- tors were cotransfected into HeLa cells with vectors expressing t-KSRP or PTBP2. A Renilla luciferase phRL-TK vector was used as an internal control for trans- fection eﬃciency. To terminate transcription, actinomycin D (10 mM) was added 24 h after transfection. RNAs were puriﬁed by Trizol, separated on formaldehyde agarose gels, transferred to Hybond-N + membranes overnight and hybridized with a [P32]dCTP randomly labeled EGFP DNA probe at 688C for 1 h using the Quikhyb kit (Stratagene). RESULTS Pgk2 mRNA accumulates in postmeiotic germ cells Although Pgk2 mRNAs are transcribed at the onset of meiosis in male germ cells (4), PGK2 protein is ﬁrst detected many days later in late-stage postmeiotic germ cells (5). Pgk2 mRNA levels increase as the germ cells diﬀerentiate (4), and northern blotting and microarrays have revealed a marked increase in Pgk2 mRNA levels in the testes of adult mice compared to prepuberal mice (3,5). To quantify Pgk2 mRNA levels in prepuberal and adult mice, semiquantitative RT–PCR was used. An increase of Pgk2 mRNA levels of at least 20-fold was detected, con- sistent with previous reports (Figure 1A) (3,5). To com- pare Pgk2 mRNA transcription in prepuberal and adult testes, nuclear run-on assays were conducted. RNA was transcribed from isolated testicular nuclei, puriﬁed and measured by hybridization to membrane-ﬁxed DNA. DNAs encoding transition protein 2 and glyceraldehyde phosphate dehydrogenase served as control transcripts for postmeiotic and constitutive transcription, respectively