Journal: The Journal of biological chemistry
Article Title: The GPCR adaptor protein Norbin controls the trafficking of C5aR1 and CXCR4 in mouse neutrophils.
doi: 10.1016/j.jbc.2024.107940
Figure Lengend Snippet: Figure 6. Norbin is required for sustained b-arrestin recruitment and promotes the agonist-induced internalization of C5aR1 in a b-arrestin- dependent manner. A, b-arrestin recruitment. Purified Ncdnfl/fl(black) and NcdnDmye (red) neutrophils were incubated for 10 min at 37 C while being stimulated with 15 nM C5a for 0, 0.17, 1 or 10 min, as indicated. Cells were lysed, a post-granule supernatant (PGS) was prepared by centrifugation, and glycosylated proteins, including C5aR1, were purified from the PGS using wheat-germ agglutinin agarose. Proteins were analyzed by SDS-PAGE and western blotting with C5aR1 and b-arrestin (b-Arr) antibodies. Representative western blots are shown. Blots were quantified by Fiji densitometry, and the b-arrestin signal was normalized to the amount of C5aR1 in each lane. Data are mean ± SEM of five independent experiments; each dot is the mean of one experiment. Statistics are two-way ANOVA with Sidák’s multiple comparisons test. p-values in black show significant differences, p-values in gray are not significant. B, b-arrestin-dependent agonist-induced internalization. Bone marrow cells were pretreated with 100 mM barbadin for 30 min at 37 C (filled symbols), or mock-treated with 1% DMSO (open symbols), and stimulated for 5 min with 50 nM C5a, or mock-stimulated (control). Cells were stained on ice to identify live neutrophils (Mac1hi, Ly6Ghi, FVDlo) and quantify the level of C5aR1 on the neutrophil surface by flow cytometry. The mfiof C5aR1 and Mac1 on the neutrophil surface were analyzed using FlowJo. Data are mean ± SEM of 3 independent experiments; each dot is the mean of one experiment. Statistics are two-way ANOVA with Sidák’s multiple comparisons test. p-values in black show significant differences, p-values in gray are not significant. C, barbadin blocks the recruitment of the clathrin-adaptor AP2. Purified Ncdnfl/flneutrophils were incubated for 30 min at 37 C in the presence of 100 mM barbadin or mock-treated, prior to stimulation with 15 nM C5a for 1 min at 37 C. Glycosylated proteins purified from the PGS using wheat-germ agglutinin agarose as in (A) and analyzed by western blotting with AP2a1 antibody. Coomassie staining was used as a loading control. The blot shown is representative of two independent experiments. D, receptor degradation. Purified neutrophils were incubated for 2 h at 37 C while being stimulated with 15 nM C5a for the indicated periods of time towards the end. Cells were fixed, permeabilized, and stained to analyse the total cellular level of C5aR1 by flow cytometry. Left-hand panel: The mfiof total neutrophil C5aR1 was analyzed using FlowJo. Right-hand panel: AUC of the 2 h time course. Data are mean ± SEM of three independent experiments; each dot is the mean of one experiment. Statistics are two-tailed paired Student’s t test. p-values in gray are not significant.
Article Snippet: Beads were washed 3 × with 1 ml lysis buffer, and proteins were eluted by incubation in 1.3 × SDS-PAGE sample buffer at 50 C for 20 min. Proteins were analyzed by SDS-PAGE and western blotting with C5aR1, b-arrestin 1/2 (Cell Signaling Technologies, 4674, 1:500) and AP2a1 (Proteintech, 29887-1- AP, 1:1000) antibodies.
Techniques: Incubation, Centrifugation, SDS Page, Western Blot, Control, Staining, Cytometry, Two Tailed Test
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