Anti myh9

OS cell lysates were prepared using the Mammalian Protein Extraction Kit (CWBIO, Beijing, China), the protein concentration was tested using the Bicinchoninic Acid (BCA) Protein Assay Kit (CWBIO). Then, the protein was separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% nonfat milk powder, the membranes were incubated with primary antibody including anti-MYH9 (#sc-98978, Santa Cruz Biotechnology, Dallas, Texas, USA), anti-β-catenin (#610154, BD Biosciences, San Jose, CA, USA), anti-c-Myc (#ab40798, Abcam, Cambridge, UK), anti-cyclin D1 (#ab226977, Abcam), anti-GAPDH (#sc-32233, Santa Cruz Biotechnology) and anti-Mouse/Rabbit IgG H&L (HRP) secondary antibody. The positive protein signal was visualized with the Immobilon-Western HRP kit (Millipore) and exposed to X-ray films.

On-bead target pull-down and gel running was performed as described above and the proteins were transferred to a nitrocellulose membrane. The membrane was then blotted and visualized using the ScanLater Western Blot Kit (Molecular Devices, Sunnyvale, CA) as follows: the membrane is blocked for 1 hour in the blocking buffer, then, separately, 1 µg/ml anti-plectin (ab83497, Abcam), 1:100 anti-MYH9 (sc-98978, Santa Cruz Biotech, Santa Cruz, CA), or 1:100 anti-AHNAK (sc-98373) was added and incubated for 2 hours to overnight. After 5 minutes, the membrane is washed with the wash buffer and is incubated in 1:5000 Eu-labelled anti-rabbit antibody in 1x blocking buffer for 1 hour. The membrane is then washed, and visualized using the SpectraMax i3 spectrophotometer (Molecular Devices). The protein ladder used was Precision Plus Protein Dual Color ladder (Bio-Rad).