Anti eef1a1

Western blots were carried out as described previously (Ventoso et al., 2006 (link)) using the following primary antibodies: anti-RPS4X (sc-85133, Santa Cruz Biotech.), anti-RPS7 (sc-377317, Santa Cruz Biotech.), anti-RPS6 (sc-74576, Santa Cruz Biotech.), anti-eIF4A (STJ2724, St. John´s lab,), anti-eIF3g (STJ23512, St. John´s lab), anti-EGFP (11814460001, Roche), anti-eEF1A1 (2551, Cell Signaling), anti-AKT1 (9272, Cell Signaling), anti-ACTB (T-5168, Sigma), anti-eIF4b (sc-376062, Santa Cruz Biotech.), anti-HRas (sc-53959, Santa Cruz Biotech.), anti-CCND3 (sc-453, Santa Cruz Biotech.), anti-ODC1 (sc-398116, Santa Cruz Biotech.), anti-GRK2 (a gift from C. Murga, CBMSO). Blots were developed with ECL (GE) and bands were quantified by densitometry. Immunofluorescence analysis was carried out as described previously using anti-RPS7 (1:500) and anti-mouse Alexa 595 as secondary antibody (Invitrogen). The preparations were analyzed under a Nikon A1R confocal microscope.

RIP assay was performed using the RNA Immunoprecipitation Kit (Geneseed, Guangzhou, China). Briefly, anti-EEF1A1 (Cell Signaling Technology, MA, USA) or anti-IgG (CST) antibody was captured with magnetic beads and incubated with total RNA lysate of AGS cells. Finally, the protein-binding RNA was extracted and purified, and detected by qRT-PCR.