Antipain

Activation levels of endogenous Rac were assayed in cells infected with ARNO-expressing adenoviruses as described above. Endogenous GTP-Rac was isolated by incubation with a GST fusion to the PBD domain of PAK and quantitated as described previously (Criss et al., 2001 (link)).
Activation of endogenous ARFs by ARNO expression was assayed using a novel pulldown assay. Cells were infected with ARNO-expressing adenoviruses in serum-free DME in the presence or absence of doxycycline as described above for 2 h. 20 ng/ml doxycycline was then added and the cells were incubated for an additional hour. Extensive ruffling and migration of the ARNO-expressing cells was apparent at this point. Cells were then lysed at 4°C in 0.65 ml of 50 mM Tris, pH 7.5, 100 mM NaCl, 2 mM MgCl₂, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10% glycerol with 0.1 mM PMSF and 1 μg/ml each pepstatin, leupeptin, and antipain. Lysates were clarified by centrifugation at 16 g for 2 min in the presence of CL-4B Sepharose beads (Amersham Pharmacia Biotech). 0.5 ml of the clarified lysate was incubated with 40 μg of GST-GGA3 bound to glutathione-Sepharose beads (Amersham Pharmacia Biotech) for 30 min. The beads were then washed three times with 50 mM Tris, pH 7.5, 100 mM NaCl, 2 mM MgCl₂, 1% NP-40, 10% glycerol with 0.1 mM PMSF, and 1 μg/ml each of pepstatin, leupeptin, and antipain. Bound proteins were eluted into 60 μl SDS-PAGE sample buffer. This sample was divided (15 μl for ARF1 and 40 μl for ARF6) and assayed for the presence of endogenous ARF1 and ARF6 by Western blotting with ARF1- and ARF6-specific antibodies. Total levels of each ARF in the starting lysates were assayed by Western blotting of 3% of the clarified lysate.

M. xanthus strains were cultured in CYE [1% (w/v) Bacto Casitone peptone, 0.5% (w/v) yeast extract, 0.1% (w/v) MgCl₂, and 10 mM Mops (pH 7.4)] broth with shaking (220 rpm) or on CYE solidified with 1.5% agar, at 32°C. To examine the effects of protease inhibition on CglB liberation, cells were grown in the presence of individual protease inhibitor panel constituents (Sigma-Aldrich, catalog no. INHIB1) at the recommended concentration: 4-(2-aminoethyl benzenesulfonyl fluoride HCl (1 mM), ε-aminocaproic acid (5 mg/ml), antipain HCl (100 μM), aprotinin (300 nM), benzamidine HCl hydrate (2 mM), bestatin HCl (40 μM), chymostatin (50 μg/ml), E-64 (10 μM), EDTA (1 mM), N-ethylmaleimide (500 μM), leupeptin hemisulfate (75 μM), pepstatin A (1 μM), phosphoramidon disodium salt (10 μM), and soybean trypsin inhibitor (1 μM). Cell resuspensions were done in TPM buffer [10 mM tris-HCl (pH 7.6), 8 mM MgSO₄, and 1 mM KH₂PO₄]. All M. xanthus and E. coli strains used are listed in tables S2 and S3 (respectively). All plasmids used are listed in table S4.
For vancomycin-susceptibility testing, 3 ml of CYE broth (in sterile 10-ml glass tubes) were inoculated to a starting OD₆₀₀ (optical density at 600 nm) of 0.05 in the absence/presence of EDTA (1 mM), with vancomycin added at increasing concentrations (0 to 100 μg/ml). Tubes were incubated with shaking (220 rpm) at 32°C for 26 hours, followed by mixing via vortex and aspiration and ejection using a pipette to break up aggregates; 1 ml of culture was then used to read the OD₆₀₀ via spectrophotometer in a disposable cuvette.

Exocyst complex purification was performed as described previously (Rossi et al., 2020 (link)). In brief, yeast strains containing a chromosomal copy of C-terminally-tagged Sec8-3xMYC and plasmids expressing wild-type Exo84 or Exo70, dominant mutations of Exo84, or the dominant Exo70-I114F mutant as the sole source of Exo84 or Exo70, respectively, were grown overnight at 30°C in synthetic media to mid-log phase (OD₅₉₉ 1.5). Cells were shifted to YPD (2% glucose) for 2 h to a final OD₅₉₉ of 3.0. To kill the cells, sodium azide and sodium fluoride were added to 20 mM final, then cells were spun at 6,700 × g_max in a JLA10.5 rotor for 6 min and washed with cold 10:20:20 buffer (10 mM Tris, pH 7.5, 20 mM NaN₃, and 20 mM NaF) before freezing on dry ice. Approximately 50 g of cells were lysed in a bead beater in lysis buffer (20 mM Pipes, pH 6.8, 120 mM NaCl, 1 mM EDTA, and 1 mM DTT) with protease inhibitors (2 μg/ml leupeptin, 2 μg/ml aprotinin, 2 μg/ml antipain, 14 μg/ml pepstatin A, 2 mM 4-[2-aminoethyl] benzene-sulfonyl fluoride, and HCl). Lysates were cleared by centrifugation at 17,418 × g_max for 12 min at 4°C in a JA25.5 rotor, and the supernatant was removed and spun again at 50,000 × g_max for 30 min in a 41Ti rotor. The final supernatant concentration was adjusted to 30 mg/ml by Bradford assay before preclearing with Sepharose beads for 1 h at 4°C to reduce nonspecific binding. The lysate was spun for 5 min to remove the Sepharose beads and incubated overnight on ice with 9E10 monoclonal anti-myc antibody. Protein A Sepharose beads were added for 2 h at 4°C. The beads were washed three times in lysis buffer and then two times in cleavage buffer (20 mM Tris, pH 7.4, 140 mM NaCl, 0.1 mM EDTA, and 1 mM DTT) before cleaving in 1 ml of cleavage buffer with TEV enzyme for 4 h at 17°C. After removal of the Protein A Sepharose beads, the cleaved exocyst complexes were collected, aliquoted, and frozen at −80°C.