Eif4g sc 11373

HeLa cells were seeded on 8-well glass slides (Millipore). Cells were transfected 24 hours after seeding using FuGene 6 (Promega) with mCherry-UBQLN2-Full-Length (wild-type), mCherry- UBQLN2-ΔUBA, mCherry-UBQLN2–450-624, or mCherry-UBQLN2–487-624 construct. 24 hr post transfection, cells were stressed with 500 µM sodium arsenite (Sigma-Aldrich) for 30 min. Cells were then fixed with 4% paraformaldehyde (Electron Microscopy Sciences), permeabilized with 0.5% Triton X-100, and blocked in 5% bovine serum albumin (BSA). Primary antibody used was against eIF4G (sc-11373; Santa Cruz). For visualization, the appropriate host-specific Alexa Fluor 488 (Molecular Probes) secondary antibody was used. Slides were mounted using Prolong Gold Antifade Reagent with DAPI (Life Technologies). Images were captured using a Leica TCS SP8 STED 3X confocal microscope (Leica Biosystems) with a 63x objective. Three independent experiments were performed.

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.