Anti gemin5

HEK293 cells (4× P100), grown in Dulbecco's Modified Eagle Medium (DMEM), transfected with the plasmids expressing p85-wt-TAP or p85-A951E-TAP proteins, were harvested 24 h post-transfection. The complexes associated to the TAP-tagged constructs were purified as described (33 (link)). Briefly, the supernatant of the first IgG Sepharose purification was subsequently subjected to a second Calmodulin (Agilent Technologies) purification step. Purified proteins were precipitated with 10% trichloroacetic acid at 4°C overnight, pelleted at 14 000 g for 15 min at 4°C, washed three times with 1 ml of acetone and dissolved in SDS-loading buffer. An aliquot (25%) was analyzed on silver stained SDS-PAGE gels to visualize the purification of proteins associated to Gemin5 p85-wt-TAP or p85-A951E-TAP polypeptides. Immunodetection of Gemin5 and p85 was performed using anti-Gemin5 (Novus) antibody.

Gemin5 protein or its tagged-domains were immunodetected using anti-Gemin5 (Novus) antibody. P0 was detected with the monoclonal antibody 3BH5 (46 (link)). Commercial antibodies were used to detect eIF3b, eIF4B, DHX9 (Bethyl laboratories), His-tag, tubulin (Sigma), PTB (Acris), hnRNPA1, hnRNPU (Immuquest), CBP (Abcam), GST, RACK1, RPL3, RPL4 (Santa Cruz), eIF4E (Transduction laboratories). Appropriate secondary antibodies (Thermo Scientific) were used according to the manufacturer instructions. Protein signals were visualized with ECL plus (Millipore). Quantification of the signal detected was done in the linear range of the antibodies.