Hierarchical Organization of Multi-Site Phosphorylation at the CXCR4 C Terminus
Figure 1
Specificity of phospho-sensitive anti-CXCR4 antibodies.
A, Schematic representation of the anti-pS324/325, anti-pS338/339, anti-pS346/347, and anti-S341-S352 (UMB-2) antibodies with their epitopes in the human CXCR4 receptor. B–G, HEK293 cells stably transfected with hemagglutinin (HA) epitope-tagged CXCR4 received CXCL12 (20 nM) or vehicle for 15 min and were lysed. An aliquot of the lysate from stimulated cells was dephosphorylated using Lambda-Protein Phosphatase (λ-PP). Samples were separated on 10% SDS polyacrylamide gels, blotted, and detected with anti-S341-S352 (B), anti-pS346/347 (C), anti-pS338/339 (D), anti-pS324/325 (E), anti-HA-tag (F), and anti-transferrin receptor (TFR) (G). B, CXCL12 stimulation causes a strong loss of anti-S341-S352 binding; dephosphorylation restores antibody binding. C–E, The phospho-selective antibodies recognize CXCR4 only in the non-dephosphorylated sample from stimulated cells. Anti-pS324/325 produces a non-specific band at approximately 70 kDa (arrowhead in E). H, HEK293 cells transiently transfected with a HA-CXCR4-ST/A mutant (all serines and threonines in the C-terminal domain were converted into alanines) were stimulated with CXCL12. Four aliquots were loaded in a Western blot and separately detected with the indicated antibodies. The phospho-selective antibodies do not detect the CXCL12-stimulated phosphorylation-deficient CXCR4 mutant (the arrow points to 47 kDa which corresponds to the size of CXCR4). F,G,H, Equal loading was controlled in stripped blots by detecting the HA-tag of recombinant expressed CXCR4 and by detecting endogenous TFR.