Optimization of immunoprecipitation of endogenous IGF2BP3. (A,B) Eluates from the immunoprecipitation reactions of IGF2BP3 from 25 mln HCT116 cells (∼2.5 mg of total protein) with Proteintech (14642-1-AP) anti-IGF2BP3 antibody coupled to protein G Dynabeads at indicated amounts resolved on SDS–PAGE and stained with a Colloidal Coomassie. * IgG heavy chain. (C) Western blot of the IGF2BP2 and IGF2BP3 CRISPR–Cas9 knockout HCT116 treated with the siRNA against IGF2BP3 and IGF2BP2, respectively, to deplete the remaining paralog. (D) Mass spectrometry intensities in the eluate of the immunoprecipitation of IGF2BP3 with Proteintech (14642-1-AP) anti-IGF2BP3 antibody. (E) Western blot of the IP described in (B). The input, unbound fraction, and eluates were loaded at a 1:1:1 ratio.
