Journal: Frontiers in Immunology

Article Title: CRBN Is a Negative Regulator of Bactericidal Activity and Autophagy Activation Through Inhibiting the Ubiquitination of ECSIT and BECN1

doi: 10.3389/fimmu.2019.02203

Figure Lengend Snippet: CRBN inhibits the ubiquitination of BECN1. (A) HEK293T cells were transfected with Mock, Flag-BECN1, HA-Ub, or Myc-TRAF6, as indicated. At 38 h after transfection, transfected cells were extracted, immunoprecipitated with anti-Flag antibody, and then an IB assay was performed with anti-Flag, anti-Myc, or anti-HA antibody. (B) HEK293T cells were transfected with Mock, Flag-BECN1, HA-Ub, Myc-TRAF6, or different concentrations of Myc-CRBN, as indicated. At 38 h after transfection, transfected cells were extracted, immunoprecipitated with anti-Flag antibody, and then an IB assay was performed with anti-Flag, anti-Myc, or anti-HA antibody. (C) Control (Ctrl) and CRBN KD THP-1 cells were treated with or without LPS (200 ng/ml) for different times, as indicated. Cells were lysed and subjected to SDS-PAGE, followed by immunoblotting with LC3-I/-II or GAPDH antibody. Band intensity was quantified using Image J software. Quantitative data were calculated from 3 independent experiments. Data are shown as mean ± SEM. (D) Ctrl and CRBN KD THP-1 cells were treated with or without LPS (200 ng/ml) for 6 h, and then fixed. Immunofluorescence assay was performed with anti-LC3 antibody. Digital images were captured with confocal microscopy, and the number of LC3-puncta was scored. Quantification represents the mean ± SEM of puncta per cell ( n = 5) from 3 independent experiments. Scale bar: 10 μm.

Article Snippet: Flag-tagged TRAF6 (Ca# 21624), Flag-tagged CRBN (Ca# 107380), and Flag-tagged BECN1 (Ca# 24388) were purchased from Addgene, Cambridge, MA, USA.

Techniques: Transfection, Immunoprecipitation, SDS Page, Western Blot, Software, Immunofluorescence, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: CRBN Is a Negative Regulator of Bactericidal Activity and Autophagy Activation Through Inhibiting the Ubiquitination of ECSIT and BECN1

doi: 10.3389/fimmu.2019.02203

Figure Lengend Snippet: CRBN is negatively involved in mROS generation and autophagy activation in response to TLR4 stimulation. Engagement of TLR4 ligand leads to concomitant trafficking of TRAF6 to mitochondria, where TRAF6 interacts with and ubiquitinates ECSIT. ECSIT ubiquitination alters OXPHOS activity, induces the generation of mROS, and that is implicated in bactericidal activity (indicated as arrow A ). However, if CRBN interacts with TRAF6, the interaction interrupts the association of TRAF6-ECSIT, and inhibits the ubiquitination of ECSIT (indicated as arrow B ). Upon TLR4 stimulation, TRAF6 interacts with BECN1, and induces ubiquitination of BECN1. Ubiquitinated BECN1 is implicated in the activation of autophagy (indicated as arrow C ). In contrast, the interaction between CRBN and TRAF6 inhibits TRAF6 E3 ligase activity and that leads to the inhibition of BECN1 ubiquitination, and results in the suppression of autophagy activation (indicated as arrow D ).

Article Snippet: Flag-tagged TRAF6 (Ca# 21624), Flag-tagged CRBN (Ca# 107380), and Flag-tagged BECN1 (Ca# 24388) were purchased from Addgene, Cambridge, MA, USA.

Techniques: Activation Assay, Activity Assay, Inhibition