Fus antibody pa5 52610

For RIP assay, caput and cauda SPZ collected from CTRL, HFD and FS mice (n=6 samples for each experimental group) and cauda SPZ of CTRL mice in vitro treated with vehicle or H₂O₂ (n=6 samples for each experimental group) were lyzed in 500 µl of RIP lysis buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 5 mM EDTA; 1% NP-40; 0.1% SDS) supplemented with RNase inhibitors (100 U/ml) and protease inhibitors (10 μg/ml of leupeptin, aprotinin, pepstatin A, chymostatin, and 5 μg/ml of TPCK). A concentration of 500 µg of each lysate was incubated with 5 µg of FUS antibody (PA5-52610; Invitrogen, Milano, Italy) or IgG (12370; Sigma-Aldrich, Milano, Italy) under rotary agitation at 4°C overnight. Protein A/G PLUS Agarose Beads (sc-2003; Santa Cruz Biotechnology, Heidelberg, Germany) were added to each sample and incubated at 4°C for 4 h. Four washes with cold TBS pH 7.6, at 3000 × g for 5 min at 4°C, were conducted and then bead pellets were resuspended in 500 µl of Trizol Reagent (Invitrogen Life Technologies, Paisley, UK) to isolate RNA, following the manufacturer’s instructions. The immunoprecipitated RNAs with FUS and control IgG was quantized (ng/µl) using a NanoDrop 2000 spectrophotometer (Thermo, Waltham, MA, United States) and used for circMEMO1 and circMAPT qRT-PCR analysis, using specific primers.

Caput/cauda epididymis and SPZ collected from CTRL and HFD male mice (n = 3 samples for each experimental group) were used for RIP experiments. All samples were lysed in RIPA buffer to obtain total lysates as reported above. Then, 5 µg of FUS antibody (PA5-52610; Invitrogen, Milano, Italy) or IgG (12,370; Sigma-Aldrich, Milano, Italy) was incubated at 4 °C overnight under rotary agitation with an equal concentration of protein lysate. Each sample was then incubated for 4 h at 4 °C with an opportune volume (60 µL) of protein A/G PLUS agarose beads (sc-2003; Santa Cruz Biotechnology, Heidelberg, Germany). After washing with cold TBS pH 7.6, bead pellets were used to carry out total RNA extraction by using TRIzol Reagent (Invitrogen Life Technologies, Paisley, UK) as reported above. Finally, a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify the immunoprecipitated RNAs with FUS and control IgG. The immunoprecipitated RNAs were stored at −80 °C until circADAM10 and circPCSK6 qRT-PCR analysis.