Anti α tubulin b 5 1 2

Cells were lysed in lysis buffer [50 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 5 mmol/L EGTA pH 8.0, 5 mmol/L EDTA pH 8.0, 1% (v/v) NP40, 0.1% (w/v) SDS, 0.1% (w/v) sodium deoxycholate, phosphatase inhibitor cocktail (25 mmol/L β-glycerophosphate, 50 mmol/L NaF, 5 mmol/L Na₂MoO₄, 0.2 mmol/L Na₃VO₄, 5 mmol/L EDTA, 0.5 μm microcystin], complete EDTA-free protease inhibitor cocktail (Roche). A total of 8% or 10% SDS polyacrylamide gels were prepared and proteins were separated followed by the transfer to nitrocellulose membranes. The following antibodies were used: anti-α-tubulin (B-5-1-2, 1:1,000, Santa Cruz Biotechnology, #sc-23948, RRID:AB_628410), anti-β-actin (AC-15, 1:10,000, Sigma-Aldrich, #A5441, RRID:AB_476744), anti-chTOG (H-4, 1:400, Santa Cruz Biotechnology, #sc-374394, RRID:AB_10987687), anti-Plk4 (6H5, 1:400, Merck Millipore, #MABC544, RRID:AB_2893410), anti-STIL (1:5,000, Bethyl Laboratories, #A302442A, RRID:AB_1944269), anti-HER2 (1:1,000, Cell Signaling Technology, #2242, RRID:AB_331015), anti-mouse/rabbit-HRP (1:10,000, Jackson ImmunoResearch, #115-035-146, #111-035-144, RID:AB_2307392). All Western blots were performed from at least three independent experiments.

Cells (1 × 10⁵) were washed once with PBS (without calcium), lysed in 100 μl of SDS-loading buffer (100 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol, 2% ß-mercaptoethanol) and sonicated for 5 min. The samples were boiled for 5 min and then subjected to SDS-PAGE. The separated proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA), which was then incubated with 1: 1,000-diluted primary antibody and then with HRP-conjugated anti-mouse or anti-rabbit antibody (Cell Signaling Technology) as recommended by the manufacturers. Primary antibody binding was detected using a Clarity Western ECL substrate (Bio Rad, Hercules, CA), and images were captured with a CCD camera (Fuji Film, Tokyo, Japan). The following primary antibodies were used: anti-phospho-p44/42 MAPK (ERK1/2, Thr202/Tyr204) (20G11, Cell Signaling Technologies, Danvers, MA, USA), anti-ERK (137E5, Cell Signaling), anti-phospho-Akt (T308) and anti-Akt pan (C31E5E and C67E7, Cell Signaling), anti-phospho-NF-κB p65 (S536) and anti-NF-κB p65 (93H1 and D14E12, Cell Signaling), anti-IκBα (L35A5, Cell Signaling), anti-pIκBα (Ser32, 14D4, Cell Signaling) and anti-α-tubulin (B-5-1-2, Santa Cruz Biotechnology, Dallas, TX, USA).

Cells (1×10⁵) were washed once with PBS, lysed in 100 μl SDS loading buffer (100 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol, and 2% β-mercaptoethanol) and sonicated for 5 min. The samples were boiled for 5 min and subjected to SDS-PAGE, and the separated proteins were transferred onto a PVDF membrane (Merck Millipore) that had been incubated with 1:1,000-diluted primary antibody and then with HRP-conjugated anti-mouse or anti-rabbit antibody (Cell Signaling), as recommended by the manufacturers. Primary antibody binding was detected using a Clarity™ Western ECL substrate (Bio Rad, Hercules, CA, USA), and images were captured by a CCD camera (Fuji Film, Tokyo, Japan). The following primary antibodies were used: anti-pERK (20G11, Cell Signaling), anti-ERK (137E5, Cell Signaling), anti-phospho-Akt (Thr308) (C31E5E, Cell Signaling), anti-Akt pan (C67E7, Cell Signaling), anti-phospho-FAK (Tyr925) (Cell Signaling), anti-FAK (Cell Signaling), anti-phospho-NF-κB p65 (Ser536) (93H1, Cell Signaling), anti-NF-κB p65 (D14E12, Cell Signaling), and anti-α-tubulin (B-5-1-2, Santa Cruz Biotechnology, Dallas, TX, USA).