huprot human proteome microarray v3.0  (CDI Laboratories)

Journal: iScience

Article Title: Stress-induced NEDDylation promotes cytosolic protein aggregation through HDAC6 in a p62-dependent manner

doi: 10.1016/j.isci.2021.102146

Figure Lengend Snippet: Identification of HDAC6 as a novel NEDD8-binding partner (A) Schematic illustration of strategy for profiling the NEDD8-binding proteins using a protein microarray (left panel). Total 17 proteins containing NEDD8 were identified as a NEDD8 binding proteins (right panel). (B) HeLa cells were transfected with mCherry-NEDD8 and GFP-HDAC6, and the colocalization of two proteins was analyzed in the absence or presence of MG132 (5 μM). Enlarge insets represent the NEDD8/HDAC6-colocalized structure observed in perinuclear region upon MG132 treatment. Nuclei were stained with DAPI (blue). DMSO was used as a negative control. (C) Recombinant GST and GST-HDAC6 were loaded in 8%–16% SDS-PAGE and visualized by staining with Coomassie Brilliant Blue (left panel). Dot blot assay was performed with recombinant NEDD8 and HDAC6 as indicated. NEDD8-binding activity to HDAC6 was detected by anti-NEDD8 antibody (right panel). GST was used as a negative control. (D) The kinetic interaction of NEDD8 to HDAC6 was monitored at pH 4.0 using the Octet QKe System (ForteBio) as describe in Transparent Methods. See also Figure S1 .

Article Snippet: To address this, we utilized the HuProt v3.0 human proteome microarray (CDI Laboratories, USA), which contain over 21,000 human proteins, to identify proteins that bind directly to NEDD8.

Techniques: Binding Assay, Microarray, Transfection, Staining, Negative Control, Recombinant, SDS Page, Dot Blot, Activity Assay