Anti ha 11 clone 16b12

Cells were lysed in lysis buffer (50 mM Tris-HCl -pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton-X, 1mM DTT, 1 mM PMSF, and supplemented with complete protease inhibitor mix (Roche),) for 30 min on ice, followed by centrifugation at maximum speed for 10 min at 4°C. An equal volume of 4x Laemmli buffer was added to the supernatants and the samples were boiled for 5 min. Approximately 1x10 5 cells were separated on an SDS-PAGE gel, followed by blotting to nitrocellulose membranes. The membranes were blocked in 5% BSA in TBST, incubated with primary antibodies, followed by an incubation with horseradish peroxidase (HRP) secondary antibodies (Mouse IgG HRP Linked Whole Ab-GE Healthcare, #NA931, 1:5000; Rabbit IgG HRP Linked Whole Ab-GE Healthcare, #NA934, 1:5000) in 3% milk in TBST. Primary antibodies: M2 Flag (Sigma Aldrich, 1:10,000 dilution), anti-c-Myc antibody clone 9E10 (Millipore, 1:1000 dilution), anti-HA.11 clone 16B12 (Biolegend, previously Covance Catalog# MMS-101P, 1:1000 dilution) and gamma-tubulin clone GTU-88 (Sigma Aldrich, 1:5000 dilution). Streptavidin-HRP (Invitrogen, #1953050) was used for detection of biotinylated proteins.