Anti mouse hrp na931v

HCC1954 (ATCC), 786-0 (NCI-60), and ACHN (NCI-60) cells were cultured in RPMI-1640 media supplemented with 10% heat-inactivated FBS, 50 U/ml of penicillin, and 50 μg/ml of streptomycin. HeLa cells (ATCC) were cultured in MEM supplemented with Earle's salts, 2 mM glutamine, 10% heat-inactivated FBS, 1.5 g/l sodium bicarbonate, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 50 U/ml of penicillin, and 50 μg/ml of streptomycin. The DDK inhibitor, PHA-767491, was synthesized as described previously [34] (link). Etoposide (#341205) was from EMD Millipore. The antibodies were purchased as indicated: CST: PARP (#9542), LATS1 (#3477), MST1 (#3682), MST2 (#3952), SAV1 (#13301), YAP/TAZ (#8418), pYAP S127 (#13008); Bethyl Laboratories Inc.: pMCM2 S53 (A300-756A), MCM2 (A300-122A), LATS2/LATS1 (A300-479A); MBL International Corporation: CDC7 (K0070-3S); Sigma: β-actin (A5441); GE Healthcare: anti-mouse-HRP (NA931V), and anti-rabbit-HRP (NA934V).

HEK 293T TetBxb1BFP cells were transfected with the pCAG–NLS–HA–Bxb1 expression vector and attB–Ub3kGiM plasmids encoding the indicated ubiquitin-fused EGFP N-terminal variants. Two days after transfection, the cells were switched to Dox-containing media. BFP–/mCherry+ recombinant cells were obtained by flow sorting ten days after transfection, and allowed to expand for an additional seven days. These recombinant cells were then incubated with lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100, and Protease Inhibitor Cocktail (Sigma-Aldrich)) for 10 min at 4°C, and pelleted at 21 000 × g for 5 min. The supernatant was collected, and protein concentration was determined by DC Protein assay (Bio-Rad) against a standard curve of bovine serum albumin. 18 μg of protein was loaded per well of a NuPAGE 4–12% Bis–Tris gel (Invitrogen) in MOPS buffer, using Spectra Multicolor Broad Range Protein Ladder (ThermoFisher Scientific) for size comparison, and transferred to a PVDF membrane. Western blotting was performed using a 1:4000 dilution of anti-GFP antibody (11814460001; Roche), followed by detection with a 1:10 000 dilution of anti-mouse-HRP (NA931V; GE Healthcare), or a 1:5000 dilution of anti-beta-actin–HRP (ab8224; Abcam).