We followed the procedure reported previously 14 (link) with the following modifications. We started with one 15 cm dish of confluent HEK293T cells transiently overexpressing Flag tagged IGF2BP (1, or 2, or 3) or infected with lentiviral shRNA targeting IGF2BP1. Before collection, cycloheximide (CHX) was added to the media at 100 μg/ml for 7 min. The lysis buffer was formulated as 20 mM HEPES, pH 7.6, 100 mM KCl, 5 mM MgCl2, 100 μg/ml CHX, 1% Triton-X-100, freshly added 1:100 protease inhibitor (Roche), 40 U/ml SUPERasin (Ambion). The sample was then fractioned into 30 fractions, 0.5 ml per fraction, and analyzed by Gradient Station (BioCamp) equipped with ECONOUV monitor (BioRad, Hercules, CA) and Gilson FC203B fraction collector (Mandel Scientific, Guelph, Canada). Sample from each fraction was subjected to western blot analysis for Flag (A5892, Sigma-Aldrich), eIF3A (#3411, CST), eIF3B (sc-16377, Santa Cruz) and HuR (A-21277, Molecular Probes), or to qPCR analysis of MYC transcript.
For detection of endogenous IGF2BP proteins in ribosomal fractions, three 15 cm dishes of HepG2 cells at ~80% confluency were harvested as described above. Sample from each fraction was subjected to western blot analysis for IGF2BP1, IGF2BP2, and IGF2BP3.
For detection of endogenous IGF2BP proteins in ribosomal fractions, three 15 cm dishes of HepG2 cells at ~80% confluency were harvested as described above. Sample from each fraction was subjected to western blot analysis for IGF2BP1, IGF2BP2, and IGF2BP3.