FIGURE 1.

Identification of GLD4-interacting proteins. (A) At top is a diagram of RNA binding proteins (RBPs) that might tether GLD4 to 3′UTRs of RNAs. (B) Flow diagram to identify GLD4-associated proteins. U87MG cells were transduced with inducible pTRIPZ lentivirus expressing 3XFLAG-GLD4-myc, and the expression was induced with 1 µg/mL doxycycline for 48 h, followed by sequential FLAG immunoprecipitation, elution, MYC immunoprecipitation, elution, and mass spectrometry. The western at bottom shows amount of of tagged GLD4 present at each step. (C) Relative to an empty vector control (i.e., RFP), there were 95 GLD4-specific coprecipitating proteins, 37 of which were identified as RNA binding proteins by a gene ontology (GO) analysis. The western blots at bottom show coprecipitation of FMRP, FXR1, SERBP1, and G3BP1 with GLD4 in the absence or presence of RNase A. GAPDH and HuR as well as coprecipitation with nonspecific IgG serve as controls.