Protein a g plus agarose beads sc 2003

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.

Cells were washed once in PBS, and lysed in 1 ml of lysis buffer (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100), and centrifuged for 15 min at 12,000 rpm at 4 °C. The supernatant was transferred to a fresh tube, and immunoprecipitations were performed with anti-Flag affinity gel (A2220, Sigma) or anti-Myc antibody (Santa Cruz) followed by adsorption to protein A/G plus-agarose beads (sc-2003, Santa Cruz). After SDS-PAGE, the samples were transferred onto polyvinylidenedifluoride membranes (Amersham life science) and probed with a variety of antibodies. For detecting interaction of endogenous CK8 with TRAF6, HEK293 cells were lysed in 0.5 ml lysis buffer and immunoprecipitated with anti-TRAF6 antibody or control serum (Santa Cruz). After extensive washing with the lysis buffer, the immunoprecipitates were resolved by SDS–PAGE, followed by Western blot analysis using the anti-CK8 antibody.