Confocal Microscopy for CRBN Localization

Confocal microscopy assay for subcellular localization of CRBN was performed as previously described (16 (link), 22 (link), 24 (link)). Briefly, THP-1 cells were treated with or without LPS (200 ng/ml) for 30 min, and stained with MitoTracker Red, anti-CRBN antibody, and DAPI, as described (16 (link)). Cells were imaged on a Zeiss LSM 710 laser-scanning confocal microscope (Carl Zeiss, Jena, Germany). For LC3 puncta assay, cells were cultured on glass coverslips for overnight. The cells were fixed with paraformaldehyde (4%), and treated with 0.2% Triton X-100 (0.2%) for cell permeabilization in ice for 30 min. Immunofluorescence microscopy assay was performed as previously described (22 (link), 23 (link)). Slides were mounted in VECTASHIELD mounting medium (Vector Laboratories, Ca# H-1000), and examined under an LSM 710 laser-scanning confocal microscope (Carl Zeiss).