Fig. 3

Autophagic degradation of TRAF3 via the cargo receptor protein NDP52. a FLAG-HA-NDP52 was used as bait for co-immunoprecipitation LC-MS/MS from A549 cells. Hits were identified using CompPASS thresholding for high-confidence interacting proteins (HCIPs) and by inclusion of immediate subthreshold interactors on the proviso of known secondary interaction with at least two HCIPs (BioGRID). Known interactors of NDP52 were equally distributed among HCIP and subthreshold hits. b HEK293T cells were co-transfected with FLAG-TRAF3 and either empty vector (−) or myc-tagged forms of NDP52 (full-length, FL, or deletants of the N-terminal SKICH domain, ΔSKICH, or C-terminal Zinc Fingers, ΔZnF). Anti-myc immunoprecipitation (IP) was performed followed by immunoblotting. c A549 lysates were immunoprecipitated with anti-NDP52 antibody-conjugated beads or control rabbit IgG beads and immunoblotted. d A549 cells stably expressing FLAG-HA-TRAF3 were stained for indicated epitopes and analysed by confocal microscopy (scale bar = 10 µm). LC3B and NDP52 are both false-coloured green and are overlaid separately with magenta HA (TRAF3) in merge panels. Arrowheads indicate co-localising foci. Boxes correspond to zoomed insets. e A549 cells expressing FLAG-HA-TRAF3 were transfected with the indicated siRNA for 72 h and then cells were stained for confocal microscopy (scale bar = 10 µm). Arrowheads indicate dual foci of TRAF3 and LC3B, quantified on a per cell basis (means, n = 3, ±S.E.M., *P < 0.05, two-tailed t-test). f A549 cells were transfected with siCtrl or siNDP52 for 72 h and immunoblotted as shown. g A549-Cas9 control cells or pooled ΔNDP52 counterparts were immunoblotted as shown. Uncropped blots are available in Supplementary Fig. 10