Affinity Purification and RNase Treatment for Protein Interaction Analysis

For MS sequence determination, HEK 293T cells in T₇₅ flasks were transfected using FuGENE HD (Promega) with 15 μg of FL-SMCR8, C9orf72-FL, or pcDNA6/myc-His B empty vector and expanded for approximately 45 h, followed by whole cell lysate preparation by sonication using a Diagenode Bioruptor. IP and sample recovery were as previously described [75 (link), 76 ]. Treatment of samples with 25 μg/ml DNase-free RNase (Roche) and 25 μg/ml RNaseA (Qiagen) was conducted in the absence of RNase inhibitors.
For other protein extracts, tissues or cells were lysed in RIPA buffer (Sigma) with Mammalian Protease Inhibitor Cocktail and phenylmethanesulfonyl fluoride (Sigma) and homogenized by Diagenode Bioruptor. For tissues, 2 mm ziconium silicate beads (Next Advance, Inc.) were added to the tubes. Supernatants were recovered by centrifugation at 11K rpm at 4 °C for 15 min and resuspended in 3X SDS loading buffer.
For each interaction co-IP (Fig. 2), extracts from approximately 5 × 10⁶ 293T cells in T₇₅ flasks transfected with tagged SMCR8 and test protein constructs were prepared in 700 μl of lysis buffer (160 mM NaCl, 50 mM Tris, 1 mM EDTA, 0.25% NP40) containing protease and phosphatase inhibitors (Sigma), and RNasin (Qiagen) and vanadyl ribonucleoside complexes (Sigma) as required, and immunoprecipitated as previously described [75 (link), 76 ].