Na3vo4

All chemicals were purchased from Sigma (St. Louis, MO, USA) unless noted otherwise. Cells were lysed in RIPA buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 5 mM NaPPi, 50 mM NaF, 10 mM Na β-glycerophosphate) plus fresh HALT protease inhibitor cocktail (Pierce; Rockford, IL, USA) and 1 mM Na₃VO₄ (New England Biolabs; Ipswich, MA, USA). Lysates were sonicated for 15 s and centrifuged at 17 000 × g for 10 min at 4°C. Protein content in supernatant was quantified by BCA assay (Pierce). Protein extracts were reduced and denatured using NuPAGE (ThermoFisher Scientific) plus 1.25% β-mercaptoethanol. SDS-PAGE-separated proteins were transferred to nitrocellulose, and membranes were stained with Ponceau S to visually confirm even protein loading. Blots were probed with antibodies against ER (Santa Cruz Biotechnology, Dallas, TX, USA; cat.# sc-8002), vinculin, β-actin (Cell Signaling Technology; Danvers, MA, USA; cat.# 13901), and FLAG (Millipore Sigma; Burlington, MA, USA; cat.# F3165). Signal was detected using horseradish-peroxidase labeled secondary antibodies (GE Healthcare; Waukesha, WI, USA) and ECL substrates (Pierce).

All chemicals were purchased from Sigma unless otherwise noted. Cells were lysed in RIPA buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 5 mM NaPPi, 50 mM NaF, 10 mM Na β-glycerophosphate) plus fresh HALT protease inhibitor cocktail (Pierce) and 1 mM Na₃VO₄ (New England Biolabs). Frozen tumor fragments were thawed, homogenized, and lysed in the same solution. Cell/tumor lysates were sonicated at 30% power for 15 s, centrifuged at 17,000 x g for 10 min at 4°C, and protein content in supernatant was quantified by BCA Assay (Pierce). Protein extracts were reduced and denatured in LDS sample buffer (GenScript) plus 1.25% β-mercaptoethanol. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and stained with Ponceau S to visually confirm protein loading and transfer. Blots were probed with primary antibodies against ER (Santa Cruz Biotechnology; cat.# sc-8002), vinculin (Cell Signaling Technology; cat.# 13901), PR (Cell Signaling Technology; cat.#8757), poly/mono-ADP ribose (Cell Signaling Technology; cat.#83732), Rb (Cell Signaling Technology; cat.#9309), phospho-Rb_S780 (Cell Signaling Technology; cat.#9307), and FLAG (Millipore Sigma; cat.# F3165). Signal was detected with DyLight conjugated secondary antibodies (Cell Signaling Technology) using the LI-COR Odyssey system (LI-COR Biosciences).

Cells were treated as indicated in figures, then lysed in RIPA buffer [50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 5 mM NaPPi, 50 mM NaF, 10 mM β-glycerophosphate (Sigma), 1 mM Na₃VO₄ (New England Biolabs), protease inhibitor cocktail (Pierce)] on ice. Frozen patient-derived tumor samples and xenografts were also homogenized in RIPA buffer. Lysates were sonicated for 10 sec. and centrifuged at 18,000 × g for 10 min. Protein concentrations of supernatants were determined by BCA assay (Pierce). Samples were reduced and denatured by addition of 1.25% β-mercaptoethanol in NuPage sample buffer (Invitrogen). Samples were heated for 1 min. at 95°C before SDS-PAGE. Proteins were transferred to nitrocellulose membranes, which were blocked with 5% BSA/TBS-T and probed using antibodies against P-AKT_T308, P-AKT_S473, Actin, P-S6_S240/244, PARP, cleaved caspase-3, PR (Cell Signaling), and ER (Santa Cruz). Antibody binding was detected using HRP-conjugated secondary antibodies against mouse or rabbit Ig (GE Healthcare), and ECL substrate (Pierce).

Peak fractions of GFP-dynein-2_D1091-Q4307 after SEC were pooled and concentrated to 8 mg/ml. In order to lock dynein in its pre-powerstroke state, Mg.ATP (Sigma Aldrich) and Na₃VO₄ (New England Biolabs) were added to a final concentration of 3 mM each. Crystals were obtained by hanging drop vapour diffusion at 19 °C mixing equal volumes of protein with reservoir solution (4-6 % PEG 6000 and 0.1 M Tris pH 8.0). Crystallization strictly depended on the presence of both Mg.ATP and Na₃VO₄. Crystals did not form under apo, Mg.ATP or Na₃VO₄ conditions. The crystal quality was markedly improved by microseeding. Seeds were prepared by harvesting GFP-dynein-2_D1091-Q4307 crystals into 100 μl of reservoir solution followed by vortexing with a seed bead (Jena Bioscience) for 30 s. After diluting the seed stock 1:10000, crystallization was carried out by mixing equal volumes of protein and seeds in reservoir solution, followed by equilibration against reservoir as described above.