Fig. 3: Membrane anchoring of FYN-TRAF3IP2 is required to activate NF-κB signaling.

a Immunofluorescence images of 293T cells transfected with TRAF3IP2, FYN-TRAF3IP2 and FYN^G2A-TRAF3IP2 stained for TRAF3IP2. Scalebars represent 10 µm. b Outgrowth of Ba/F3 cells transduced with pMIG-FYN-TRAF3IP2 or pMIG-FYN^G2A-TRAF3IP2 after withdrawal of IL-3. n = 3 biological replicates per condition. c Western blot analysis for TRAF3IP2 on cytoplasmic and membrane fractions of Jurkat cells transduced with pMIG-TRAF3IP2, pMIG-FYN-TRAF3IP2 and pMIG-FYN^G2A-TRAF3IP2. d NF-κB GFP reporter signal intensity in Jurkat cells with empty control vector or expression of TRAF3IP2, FYN-TRAF3IP2 or FYN^G2A-TRAF3IP2 after stimulation with an agonistic anti-CD3ε antibody or exposure to control immunoglobulin (Ig). n = 3 biological replicates per condition for Ig control, n = 4 for anti-CD3ε. p values were calculated with Tukey’s post-hoc multiple comparisons test. e Immunofluorescence images (left) of TRAF3IP2 staining on CD4⁺ T cells transduced to express TRAF3IP2, FYN-TRAF3IP2 or FYN^G2A-TRAF3IP2. Scalebars represent 5 µm. Quantification (right) of TRAF3IP2 signal intensity in the outer cell bin, expressed as fraction of signal intensity over the entire cell. Each dot represents a cell (n = 201 for TRAF3IP2, n = 192 for FYN-TRAF3IP2 and n = 179 for FYN^G2A-TRAF3IP2). Horizontal line and whiskers represent median and interquartile range respectively. p values were calculated with Games-Howell’s multiple comparisons test. f Intracellular flow cytometry for IκBa (top row) or phospho-p65 (bottom row) in CD4⁺ T cells treated with control immunoglobulin (Ig) or agonistic anti-CD3ε antibody. n = 3 biological replicates per condition. p values were calculated with Tukey’s post-hoc multiple comparisons test. All data are represented as mean ± SD unless stated otherwise.