α tubulin ab7291

Cultured cells and tumor samples were processed for western blot as previously described [10 (link)], except the membrane was incubated in 1 M DTT solution at room temperature for 5 min then washed with TBS-T three times before adding IGF-2 antibody. Phosphatase inhibitors (P2850, P5726, Millipore-Sigma, Burlington, MA, USA) were added to the lysis buffer for P-Akt. The antibodies used were IGF-2, 8H1, MA5-17096, 1:1000, ThermoFisher Scientific, Waltham, MA, USA, α-tubulin, Ab7291, 1:10,000, Abcam, Cambridge, UK, GAPDH, sc-47,724, 1:1000, Santa Cruz Biotechnology, Dallas, Texas, USA, Akt, 2938, P-Akt, 4058, 1:1000, secondary antibodies 7074 and 7076 S, 1:10,000, Cell Signaling Technology, Danvers, MA.

For western blot analysis, 1 × 10⁶ cells were lysed with RIPA buffer (08714-04, Nakalai Tesque). SDS sample buffer was added, and the mixture was incubated at 93 °C for 3 min. The extracted proteins were separated on Bollt 4–12%, Bis-Tris, 1.0 mm, Mini Protein Gel (NW04120BOX, Thermo Fisher Scientific) and blotted onto an Immobilon-P PVDF Membrane (IPVH00010, Merck) using a Mini PROTEAN Tetra Cell (Bio-Rad). The transferred membranes were incubated with the following primary antibodies: α-tubulin (ab7291, Abcam), pSMAD1/5/9 (9511, Cell Signaling Technology), pSMAD2 (3108, Cell Signaling Technology), pMAPK (4376, Cell Signaling Technology), pSTAT3 (9131, Cell Signaling Technology) and STAT3 (564533, BD Bioscience). The primary antibodies were detected with anti-rabbit IgG, HRP-linked antibodies (7074, Cell Signaling Technology) and anti-mouse IgG, HRP-linked antibodies (7076, Cell Signaling Technology), followed by detection using ECL Prime Western Blotting Detection Reagent (RPN2236, Amersham). Chemiluminescence images were acquired using the ImageQuant LAS 4000 (GE Healthcare) and Ambersham ImageQuant 800 (Cytiva) systems. Uncropped western blot images are shown in Supplementary Figs. 2 and 3.