Complete edta free protease inhibitor cocktail tablet

As above, cells were transfected with 2.5 μg of pcDNA 3.1(+) plasmids expressing either WT, T148D, or T148A crystallins or an empty vector (EV) and were seeded in six-well plates. The next day, transfected cells were incubated either in DMEM-NG + 10% FBS or serum-free DMEM-NG for 4 or 24 h. Cells transfected with EV served as an experimental control. Following incubation, cells were harvested on ice in chilled RIPA buffer (100 mm Tris pH 7.5, 3 mm EGTA, 5 mm MgCl₂, 0.5% Triton X-100, 1 mm PMSF, 1× complete EDTA-free protease inhibitor cocktail tablet; Roche Diagnostics) and subjected to immunoblot analyses.

Primers to introduce mutations in the Cus1(290–350) and Hsh49 gene fragments were designed and used as described in García-Nafría et al. (2016) (link). Glutathione hexa-histidine Cus1(290–350)p constructs were coexpressed with Hsh49p as described above except that cells were grown at 15°C after induction. After harvesting, cells were frozen, thawed, and resuspended in Pulldown buffer (20 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 5 mM 2-mercaptoethanol with one cOmplete EDTA-free protease inhibitor cocktail tablet [Roche] per 100 mL) then lysed by sonication. After centrifugation at 72,000g for 30 min at 4°C, 800 µL of supernatant was mixed with 10 µL 2 M imidazole-HCl, pH 7.4 and 30 µL Ni-NTA resin. After 2 h incubation with gentle mixing at 4°C, the resin was pelleted by centrifugation and washed twice with 300 µL Wash buffer (Pulldown buffer with 25 mM imidazole-HCl), then resuspended in 100 µL SDS-PAGE loading buffer, heated for 2 min at 90°C, and the protein released was analyzed by SDS-PAGE.

293T or eHAP1 cells were transfected with 3 μg PB1, 3 μg PB2 627E or 627K or PB2 Δ535–667, 1.5 μg PA from A/Tky/5092/91 (H5N1), and 3 μg indicated ANP32-FLAG. Three micrograms of green fluorescent protein (GFP)-FLAG was transfected in place of ANP32-FLAG to be used as a negative control. Seven hundred fifty nanograms of viral-like 76-nucleotide (nt) RNA was used in the experiment in Fig. 7d. Total DNA transfected was kept equal between samples.
Twenty-four hours after transfection, cells were washed in phosphate-buffered saline (PBS) followed by incubation on ice for 20 min in lysis buffer (50 mM Tris-HCl, pH 7.8 [Sigma], 100 mM NaCl, 50 mM KCl, and 0.5% Triton X-100 [Sigma], supplemented with cOmplete EDTA-free protease inhibitor cocktail tablet [Roche]). Samples were centrifuged to pellet cell debris, and cell lysate was collected.
FLAG-tagged proteins were immunoprecipitated using anti-FLAG M2 affinity gel (Sigma-Aldrich). Following three washes at 4°C in Tris-buffered saline (TBS) (Alfa Aesar), cell lysate was incubated with the FLAG affinity gel at 4°C on a rotator overnight. After a further three washes with TBS at 4°C, proteins were eluted by addition of 100 μl 3× FLAG peptide at 150 ng/μl. Coimmunoprecipitated proteins were detected using immunoblot analysis.

These were performed as described previously [20 (link)]. Briefly, C. albicans cultures were grown to OD₆₀₀ = 0.5, cells harvested, washed with sterile distilled H₂O and resuspended in 1 ml lysis buffer (20 mM Tris pH 7.5, 100 mM KCl, 5 mM MgCl₂, 20% glycerol, 1 mM PMSF (EMD Chemicals), 20 mM Na₂MoO₄ (Sigma), and complete EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics). Cells were then disrupted by bead beating twice for 4 minutes with 7 minute breaks on ice between cycles. Lysates were centrifuged at 13,006 g for two-times 5-minutes, recovering the supernatants at each stage. The combined lysate was then cleared by centrifugation at 21,006 g for 10 minutes at 4°C and protein concentrations determined using the Bradford assay. Anti-TAP immunoprecipitations were performed by adjusting protein concentration to 1.5 mg/ml in lysis buffer containing 0.2% Tween, and incubating with Rabbit IgG agarose (Sigma #A2909) at 4°C overnight as per the manufacturer’s specifications. Unbound material was discarded, the beads were washed five times with 1 ml lysis buffer containing 0.1% Tween, and proteins were eluted by boiling in one volume of 2.5% sample buffer (125 mM Tris-HCl, pH 6.8, 5% glycerol, 2.5% SDS, 2.5% beta-mercaptoethanol, distilled H₂O, bromophenol blue). Proteins were separated on 8% SDS-PAGE gels and probed as above.

LIS1 constructs were purified from frozen sf9 cell pellets from a 600 mL culture, as described previously^{68 (link)}. Lysis and clarification steps were similar to full-length dynein purification except lysis was performed in LIS1-lysis buffer (30 mM HEPES pH 7.4, 50 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 300 mM potassium chloride, 1 mM DTT, 0.5 mM Pefabloc, 10% (vol/vol) glycerol) supplemented with cOmplete EDTA-free protease inhibitor cocktail tablet (Roche) per 50 mL was used. The clarified supernatant was incubated with 0.5 mL of IgG Sepharose 6 Fast Flow beads (GE Healthcare Life Sciences) for 2-3 hours on a roller. The beads were transferred to a gravity flow column, washed with 20 mL of LIS1-lysis buffer, 100 mL of modified TEV buffer (10 mM Tris-HCl pH 8.0, 2 mM magnesium acetate, 150 mM potassium acetate, 1 mM EGTA, 1 mM DTT, 10% (vol/vol) glycerol) supplemented with 100 mM potassium acetate, and 50 mL of modified TEV buffer. LIS1 was cleaved from IgG beads via incubation with 0.2 mg mL^–1 TEV protease overnight on a roller. The cleaved LIS1 was filtered by centrifuging with an Ultrafree-MC VV filter (EMD Millipore) in a tabletop centrifuge. Purity was evaluated on SDS–PAGE gels, and protein aliquots were snap frozen in liquid N₂ and stored at –80 °C.