As previous reports [46 (link), 47 (link)], CM cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and quantified by the Bradford method and Ponceau S staining of nitrocellulose membranes. 30 µg protein samples were subjected to 10% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore). Immunoblot membranes were blocked in 5% milk for 1 h. Membranes were incubated with the primary antibody, including CDC42 (Santa Cruz, sc84011, 1:200), TEAD1 (Santa Cruz, sc393976, 1:200), AGO2 (Santa Cruz, sc376696, 1:500), IGF2BP2 (Proteintech, 11601-1-AP, 1:500) and GAPDH (Santa Cruz, sc-47724, 1:1000) at cold room for overnight. After three washes with TBST, membranes were incubated with secondary antibody for 1 h, washed, and exposed to Pierce ECL Western Blotting Substrate (Thermo, #32106) for detection of protein expression.
Proteins were lysed in RIPA buffer and immunoprecipitated with AGO2 or IGF2BP2 antibody and the corresponding IgG. After applying a magnet, proteins associated with Protein A/G Magnetic Beads were washed three times and analyzed by western blotting. The signal densities of protein bands were quantified and normalized to that of GAPDH using the ImageJ software.
Proteins were lysed in RIPA buffer and immunoprecipitated with AGO2 or IGF2BP2 antibody and the corresponding IgG. After applying a magnet, proteins associated with Protein A/G Magnetic Beads were washed three times and analyzed by western blotting. The signal densities of protein bands were quantified and normalized to that of GAPDH using the ImageJ software.
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