Gtpγs

Approximately 0.2 mg purified GST-tagged Rab11a or GST tag was incubated with preequilibrated glutathione Sepharose 4B beads (GE Healthcare) (100 μL slurry per 1 mL sample volume) at 4°C for 3 h in binding buffer (50 mM HEPES-NaOH [pH 7.5], 300 mM NaCl, 10% [vol/vol] glycerol, 8 mM MgCl₂, 10 μM GTPγS [Abcam]). After binding, the glutathione Sepharose beads were washed in binding buffer. Resuspended beads were then incubated in binding buffer with either 0.06 mg purified heterotrimeric polymerase, 0.08 mg purified 627-NLS or PB2-C protein, or WSN-infected or mock-infected whole-cell lysates for 1 h at 4°C. After binding, the beads were washed five times in binding buffer and resuspended in the same buffer supplemented with 1 mM DTT and 0.1 mg 3C protease. After 1 h of incubation at 4°C, targeted protein complexes were cleared from the beads by centrifugation at 1,000 × g for 5 min at 4°C. GST pulldown protein fractions were analyzed by 12% SDS-PAGE with Coomassie blue staining and/or used for viral RNA and protein analyses.

PulSA was conducted according to the previous description by Toh et al. [27 (link)]. We plated 3 × 10⁵ cells and cultured for 24 h. For RanGTP inhibition, cells were treated with 10 mM of GTPγS (Abcam) or GDPβS (Sigma) for 30 mins or 1 h, and the control groups were untreated. Cells were trypsinized and suspended in total media containing 10% fetal bovine serum albumin, washed with PBS and incubated with DCX antibody, followed by incubation in serum-free media and 4% paraformaldehyde fixation, series of washing before permeabilization in 0.1% Triton X-100, blocking with goat serum before staining with secondary antibody (FITC), and DAPI staining. Stained cells were then suspended in 1%(w/v) BSA/PBS for flow cytometry. Cells were probed in BD LSRFortessa™ flow cytometer. FCS files were exported and analyzed in Microsoft EXCELand histograms were plotted and analyzed by GraphPad prism and SPSS.

DiC8-PIP₃, In(1,3,4,5)P₄, In(1,4,5)P₃ (IP₃), and GNF362 were obtained from Cambridge Bioscience Ltd. Fura2-AM was obtained from Thermo Fisher Scientific, and U46619 was obtained from Tocris. The phospho-AKT antibody recognizing phospho-Ser⁴⁷³ (clone 11E61) was obtained from Upstate. The pan-AKT antibody (C67E7) recognizing Akt1, Akt2, and Akt3 proteins was obtained from Cell Signaling Technology. Goat antimouse antibody linked to horse-radish peroxide (HRP) and Goat antirabbit-HRP were purchased from Millipore. Antibodies to GAP1(IP4BP/RASA3), ITPKB, ITPKA, and BTK were purchased from (Santa Cruz Biotechnology Inc). The anti-Rap1(A+B) and IP₃-ELISA kit was purchased from Abcam Ltd. GTPγS and RalGDS-RBD beads were obtained from Abcam Ltd. PI(3,4,5)P₃ PIP beads (PIP₃ beads) were obtained from Echelon Biosciences (via 2B Scientific). Vena8 Cellix microfluidic chip channels were obtained from Cellix Ltd.