Eif3b sc 16377

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.

Cells were fixed with 4% (w/v) paraformaldehyde, permeabilized with 0.2% (v/v) Triton X-100 for 30 min, and blocked by 1% BSA in PBS. A monoclonal antibody against PABP-1 (10E10; Sigma-Aldrich), G3BP1 (sc-81940; Santa Cruz), eIF4G (sc-11373; Santa Cruz), and eIF3b (sc-16377; Santa Cruz) were used at a 1:500-800 dilution. The secondary antibodies were Alexa Fluor 594- or 647-labeled anti IgG of the appropriate species (anti-human antibodies were purchased from Jackson Immunoresearch, others from Invitrogen), with a 1:1000 dilution. For longer administration conditions (24 h), rapamycin was used at 100 nM. After the staining, cells were imaged with either a Zeiss Axiovert 135 TV microscope equipped with a QIclick camera (QImaging), or a Nikon eclipse Ti microscope equipped with a Zyla sCMOS camera (Andor). For imaging Alexa Fluor 594 and 647, filter sets for mCherry and Cy5 were used, respectively.