Figure 6: Rnf145 knockdown reduces ubiquitination of the Nox2 complex.
From: Functional genomics identifies negative regulatory nodes controlling phagocyte oxidative burst
(a) RAW264.7 cells were transduced with lentivirus-encoding shRNA targeting Rnf145 or control (LacZ). Cells were then left untreated or stimulated with zymosan for 30 min. Cells were then lysed, and ubiquitinated peptides were immunoprecipitated and enumerated by UbiScan (see methods). Differential abundance of each ubiquitinated peptide was compared between control and Rnf145 knockdown. The network was constructed by anchoring on hit components (>2.5-fold difference between Rnf145 knockdown and control). Hits are represented by nodes and the network was extended by iteratively incorporating interacting partners using protein–protein interaction data derived from the curated Human Protein Reference Database and genome-wide interactome screens. The network was then pruned to include only first-order interactions (represented by edges) that directly connect the hits from UbiScan (enrichment P=2 × 10−11). (b) Relative abundance of ubiquitinated peptides derived from transmembrane proteins. Values represent fold difference between Rnf145 knockdown and control. (c) Peptide sequences derived from p22phox and gp91phox showing ubiquitination sites (*), fold difference in Rnf145 knockdown relative to control and relative abundance (max intensity). (d) Knockdown of Rnf145 was performed in RAW264.7 macrophages (as in Fig. 2a), followed by stimulation with zymosan and western blot for the indicated proteins. (e) Rnf145 was expressed in RAW264.7 cells by lentiviral transduction. Cells were then lysed and analysed by western blot for gp91phox and ERK2 as a loading control. In addition, gp91phox was immunoprecipitated to detect K48-specific ubiquitin linkages by western blot. (f,g) RAW264.7 cells were transduced with lentivirus encoding Rnf145 with a C-terminal V5 epitope tag. Confocal microscopy was performed to visualize cellular localization of Rnf145 (V5), gp91phox or Derlin 1. Scale bar, 10 μm. Arrows indicate colocalization of RNF145 and Derlin 1 (f) or gp91phox-positive vesicles (g).
