Fig. 1: ACKR3 physically interacts with ACKR3. | Nature Communications

Fig. 1: ACKR3 physically interacts with ACKR3.

From: The atypical chemokine receptor 3 interacts with Connexin 43 inhibiting astrocytic gap junctional intercellular communication

Fig. 1: ACKR3 physically interacts with ACKR3.The alternative text for this image may have been generated using AI.

a Volcano plot representing proteins identified by nanoLC-MS/MS in immunoprecipitations from HEK293T cells transfected with HA-ACKR3 or empty plasmid. Proteins known to physically or functionally interact with Cx43 (GJA1) and ACKR3 are represented in orange and green, respectively. b Representative images of GFP (Ackr3), Cx43 and GFAP immunostaining in brain from 8-week-old Ackr3-EGFP BAC mice. Scale bars = 500 μm for mosaics and 100 μm for others. c Representative WB of HA-immunoprecipitations from WT or HA-ACKR3 knock-in (KI) embryonic mouse brains. d Normalized BRET values measured in HEK293T cells co-expressing the indicated proteins. ACKR3-Cx43 BRET signal variations were fitted with the one-site total binding equation and background constraint to constant value of 0 using Prism (Extra-sum-of Square, F-test, P < 0.001 vs. line through the origin, F(2,114)). e Representative WB of HA-immunoprecipitations performed from HEK293T cells expressing HA-ACKR3 or HA-CXCR4. The histogram shows the immunoreactive signals (normalized to the value in HA-ACKR3-expressing cells) of Cx43 co-immunoprecipitated with HA-receptors (Two-tailed unpaired t test, F(2,2)). f 3D reconstruction of ACKR3, CXCR4, and Cx43 immunostainings in TG1 and R633 cells. The overlapping volume represents the volume where each receptor is co-expressed with Cx43 (see Supplementary Fig. 1 for original immunoreactive signals). The histogram illustrates the percentage of Cx43 immunostaining in the overlapping volume (Two-way ANOVA, Bonferroni post-hoc, F(1,17)). Scale bar = 3 μm. g Kinetics representing normalized BRET values measured in HEK293T cells co-expressing Cx43-YFP and ACKR3-NLuc challenged with either vehicle, 10 nM CXCL12 or 100 nM CXCL11. (Two-way ANOVA, Bonferroni post-hoc, F(22,99)). h HEK293T cells expressing HA-ACKR3 challenged with vehicle, CXCL12 (10 nM) or CXCL11 (100 nM) for 30 min. Representative WB of HA-immunoprecipitations are illustrated. The histogram shows the immunoreactive signals (normalized to values from cells challenged with vehicle) of Cx43 co-immunoprecipitated with HA-ACKR3 (One-way ANOVA, Bonferroni post-hoc, F(2,12)). All data are represented as means ± SEM. See the Statistics and Reproducibility section for the number of repetitions, exact P values and symbol (*) legend. Source data and uncropped Western blots are provided as a Source Data file.

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