Pfastbachtb vector

Human Rac1 (GenBank accession no. NM_006908.4) was subcloned into pFastBacHTB vector (Invitrogen, Carlsbad, CA) and fused with an N-terminal hexa-histidine (6xHis) tag. For bacterial expression, full-length Rac1 and GDI1 (GenBank accession no. D13989) were cloned into pGEX-4T1 vector. DHPH constructs of human Vav2 (aa 168–543), human Dbl (aa 498–825), TrioN (aa 1226–1535), murine Tiam1 (aa 1033–1404), human P-Rex1 (aa 34–415), and human Pak1-GBD (aa 57–141) have been reported before [11] (link), [40] (link).

Multiple primer pairs are listed in Supplementary Table S3 to construct Flag- and hemagglutinin (HA)-tagged expression bacmids. A HA Tag and a Flag Tag within the primers at C terminus were designed for Flag-tagged and HA-tagged protein expression. The ORFs of viral proteins were amplified from the BmNPV Bacmid and cloned into the pEASY-T1 Simple vector (TransGen, Beijing, China) for sequencing. Thereafter, then ORFs were subcloned into pFastBacHTB vector (Invitrogen Life Technologies, Carlsbad, CA, USA) for the construction of recombinant bacmids according to the manufacturer’s protocol (Invitrogen Life Technologies). Tn7 cassettes from the constructed pFastBacHTB were transferred into the BmNPV bacmid to construct the recombinant viruses Bm-P47-HA, Bm-LEF4-HA, Bm-PK1-HA, Bm-LEF9-HA, Bm-IE1-HA, Bm-VLF1-HA, Bm-VLF-HA, and Bm-actin-Flag. BmN cells were transfected with 2 μg of Bm-P47-HA, or of Bm-LEF4-HA, or of Bm-LEF9-HA, or of Bm-PK1-HA, or of Bm-IE1-HA, or of Bm-VLF-HA, or of Bm-actin-Flag, using 8 μL lipoInsect (Beyotime, Shanghai, China). The cells were replenished with 2 mL of fresh cell culture medium after incubation for 5 h. Thereafter, the cells were incubated at 27 °C, and BV supernatants were collected at 96 h p.i.

Human Rac1 gene subcloned into pFastBacHTB vector (Invitrogen, Carlsbad, CA) was transformed into DH10BAC strain. Agar plates containing kanamycin, gentamycin, tetracycline, X-gal and isopropyl-β-D-thiogalactoside were used to select recombinant Rac1 clones. The Rac1-positive clones underwent two more purification steps before recombinant Rac1 bacmid were extracted. The baculoviruses (passage 1) were generated by infecting Sf9 insect cells using recombinant Rac1 bacmids. Viruses were ready to use for large scale Rac1 expression after two more amplification steps (passages 2 and 3).
Sf9 were cultured in TC-100 medium, containing 10% fetal bovine serum, penicillin, streptomycin and pluronic F-68 solution at 27°C. The titer of baculoviruses was determined by the ITCD₅₀ method [41] , [42] . The multiplicity of infection (MOI) and Rac1 expression time were optimized by infecting the Sf9 cells at different MOIs and different culture time points. Samples of infected cells (1 ml) were harvested; the cell pellets were lysed in Laemmli buffer, containing 60 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue and analyzed by immunoblotting using an anti-His-tag antibody.

Rat Syt1 cytoplasmic domain (residues 140–421, known as C2AB), C2A domain (residues 140–266), C2B domain (residues 270–421) and its mutants C2B_2RQ (residues 270–421, R398Q and R399Q), C2b (residues 270–421, D363N and D365N), full-length rat Munc18-1, full-length synaptobrevin-2 and its cytoplasmic domain (residues 29–93), rat syntaxin-1a cytoplasmic domain (residues 2–253) and its SNARE domain (H3, residues 191–253), rat Munc13-1 MUN domain (also known as MUN⁹³³, residues 933–1407, EF, 1453–1531), full-length Cricetulus griseus NSF and full-length Bos taurus α-SNAP were cloned into pGEX-KG vector. Full-length human SNAP-25a (with its four native cysteines mutated to serines), its C-terminal truncation SNAP-25a Δ9 (residues 1–197, with its four native cysteines mutated to serines) and full-length rat syntaxin-1a were cloned into pET28a vector (Novagen). The co-expressed full-length rat Munc18-1 and full-length rat syntaxin-1a, full-length rat Munc18-1 and rat syntaxin-1a cytoplasmic domain (residues 1–261) were cloned to pETDuet-1 vector (Novagen). Rat Munc13-1 C₁-C₂B-MUN fragment (residues 529–1407, EF, 1453–1531) was cloned into pFastBac™HtB vector (Invitrogen). All of the mutants used in this study were generated by using QuikChange Site-Directed Mutagenesis Kit (Stratagene).

GAK K constructs for protein purification, analog-specific (AS) and KD, were cloned into the pFastBac HT B vector (Invitrogen) by PCR cloning to contain N-terminal 6× His tag. His-tagged GAK-K-AS and GAK-K-KD baculovirus expression (Bac-to-Bac Baculovirus expression system; Invitrogen) was performed according to manufacturer's protocol in insect cells.