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. 2016 Mar 28;126(5):1664–1678. doi: 10.1172/JCI81516

Figure 7. SYTL1 promotes membrane trafficking of CXCR4 and activates downstream signaling.

Figure 7

(A) Representative immunofluorescence of H9M1, HΔM, or HΔM/SYTL1 cells after the indicated incubation periods with CXCL12 (3 experiments). Alexa Fluor 350–conjugated wheat germ agglutinin (WGA) was used to indicate plasma membrane. Scale bar: 20 μm. (B) The expression of CXCR4 on the surfaces of H9M1, HΔM, or HΔM/SYTL1 cells was analyzed by flow cytometry at the indicated times after CXCL12 stimulation. The cells were incubated with the anti-CXCR4 antibody after (same staining condition as A) or before CXCL12 stimulation. The data are representative of 3 independent experiments. (C) ERK and AKT phosphorylation after CXCL12 stimulation in the indicated cell types; blots are representative of 3 independent experiments. (D) Juxtaposition of H9M1 or HΔM/SYTL1 cells and CAR cells in bone marrow. CAR cells are stained with anti-S100. Representative images of 3 independent experiments. Quantification with statistical analysis is shown in Supplemental Figure 9D. Scale bar: 20 μm. (E) Proposed model showing enhanced membrane trafficking of CXCR4 after SYTL1 upregulation. CXCR4 is internalized and ubiquitinated upon binding to CXCL12. However, CXCR4 is rapidly provided from its reservoir on the limiting membrane of multivesicular bodies in the presence of SYTL1 and Rab27b.