Inhibition of Western Detection of DAZL1
Unlabeled DAZL1 was synthesized in vitro using the TNT Coupled Reticulocyte Lysate System (Promega). T3 and T7 RNA polymerase transcribed the Dazll cDNA clone in the sense and antisense directions, respectively.
Varying amounts of the in vitro synthesis mixtures were incubated with 2 ^l of anti-DAZL1 antiserum in a total volume of 200 ^l of TBS-T buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween-20) on ice for 1 h. The solutions were diluted to 4 ml with 10% milk in TBS-T before incubation with Western blot membranes containing equal amounts of liver and testis extracts.
Sucrose Gradient Analysis of Mouse Testicular Extracts
Mouse testicles were homogenized in either standard EB or modified EB in which 5 mM MgCl2 was replaced with 30 mM EDTA pH 7.5, or KCl was added to a final concentration of 0.5 M. After centrifugation at 10 000 X g for 10 min, 0.5 ml of PMS was layered on top of 15-45% or 5-30% sucrose gradients in 20 mM Tris pH 7.5, 0.1 M KCl, and the same concentration of MgCl2, EDTA, or KCl as in the extracts. After centrifugation in a Beckman SW41 rotor (Beckman Instruments, Palo Alto, CA) at 39 000 rpm for 2 h at 4°C, 0.5-ml fractions were collected from the bottom, and the OD254 was determined. Aliquots of every other fraction were analyzed on 10% SDS-PAGE gels and then by Western blotting with antibodies against DAZL1 and the fragile X mental retardation protein FMRP, and a human autoantibody against ribosomal P antigens (ImmunoVision, Springdale, AR). For ribonuclease (RNase) treatment, RN-ase A was added to the PMS in standard EB to a final concentration of 300 ^g/ml. After incubation at room temperature for 5 min, the solution was analyzed by sucrose gradient centrifugation as above.
Capture of DAZL1-Associated Messenger Ribonucleoprotein (mRNP) Particles on Oligo(dT) Beads
Mouse testicular PMS was subjected to mRNP capture using the Oligotex mRNA purification kit (Qiagen, Santa Clarita, CA) according to the manufacturer’s protocol. Briefly, 0.25 ml of PMS was mixed with 0.25 ml of doublestrength buffer OBB (20 mM Tris pH 7.5, 1 M NaCl, 2 mM EDTA, and 0.2% SDS) and 20 ^l of Oligotex beads. After rocking at room temperature for 10 min, the beads were spun down and washed twice with 0.5 ml of wash buffer OW2 (10 mM Tris pH 7.5, 150 mM NaCl, and 1 mM EDTA). Bound proteins were eluted by heating in 30 ^l of double-strength Laemmli sample buffer at 100°C for 5 min. Aliquots of the load, the wash, and the bound fractions were analyzed by Western blotting with antibodies against DAZL1 and FmRP Pretreatment of PMS with EDTA (30 mM, 37°C, 15 min), RNase (1.2 mg/ml, 37°C, 10 min), or NaOH (0.2 N, 37°C, 10 min, then neutralized to pH 8.0 with 1 N HCl) before capture was carried out according to Feng et al..