Pmxb10

The indicated FIP200 and RIG-I domains were cloned into pGEX-5X-3 (GE Healthcare, # 28-9545-55) to fuse with a GST tag, pET28b(+) (Novagen, # 69865-3) to fuse with a His tag, pT7-FLAG-2 (Sigma, # P1243) for a FLAG tag, or pMXB10 (New England Biolabs, # E6901S) for a MBP tag. These constructs were transfected into BL21 (DE3) E. coli (New England Biolabs, # C2527I) and cultured in LB broth at 20 °C. IPTG (0.4 mM) was added to induce protein expression. MBP-tagged proteins were purified using the IMPACT kit (New England Biolabs, # E6901S), and the MBP pull-down assays were performed using the anti-MBP Magnetic Beads New England Biolabs, # E8037S). FLAG-tagged proteins were purified by using the EZview Red Anti-FLAG M2 Affinity Gel (Sigma, # F2426). The GST Protein Interaction Pull-Down Kit (ThermoFisher Scientific, # PI21516) was used for GST-tagged protein purification and GST pull-down assays. The His-Spin Protein Miniprep kit (Zymo Research, # P2002) was used for His-tagged protein purification and His pull-down assays.

Human Tau(R406W) 0N4R form of Tau*, Tau*C2A, Tau*C2H, Tau*C291H, and Tau*S2A coding regions were cloned into pMXB10 (New England Biolabs) vector. Proteins were purified by chitin beads (NEB) as described (Venter et al., 2011 (link); Wang et al., 2010 ) and concentrations measured by the BCA kit (Thermo Scientific). In the CD assay, proteins were incubated with zinc or other metals at 37° C in 0.01 M PBS (pH = 7.4) buffer containing 1 mM dithiothreitol (DTT). CD spectra were measured in the far ultraviolet region (190–260 nm) and recorded by the Jasco J-715 Spectropolarimeter.