Figure 7

Analysis of CXCR4 Structural Heterogeneity in Neuroblastoma Cells. A) Western Blot Detection of CXCR4 in Neuroblastoma Cell Lysates. Cell lysates were generated by solubilizing neuroblastoma cells in 1% triton X-100. Detergent soluble proteins were resolved on 4-20% Bis-Tris SDS gels and transferred to nitrocellulose membranes. CXCR4 was detected by probing with a rabbit anti-human CXCR4 polyclonal primary antibody (ab2090), followed by a horse radish peroxidase conjugated donkey anti-rabbit secondary antibody, and then chemiluminescent substrate. CXCR4 was visualized using enhanced chemiluminescent (ECL). The samples represented are as follows: Lane 1- CHP-134, Lane 2-KCN, Lane 3-KCNR, Lane 4-NB-69, Lane 5-NGP, Lane 6-SK-N-FI, Lane 7-SK-N-AS, Lane 8-SK-N-ASΔ3*, Lane 9-LAN-5, Lane 10-SK-N-SH, Lane 11-SH-SY5Y. Cell lines are color coded by surface expression class: Red = Low, Blue = Medium, Green = High, Black = Not Conclusively Determined (N.D.). *SK-N-ASΔ3 is a derivative cell line of SK-N-AS that has been transfected with an expression vector containing the coding sequence for an E3 ubiquitin ligase component Cullin-5 (CUL-5). All samples were loaded equally as determined by comparing actin levels in each lane (Data not shown). B) Western Blot Detection of Ubiquitin in CXCR4 Immunoprecipitates. Cell lysates were adsorbed with rabbit anti-human CXCR4 antibodies followed by precipitation with protein G Agarose. Precipitated CXCR4 Immune complexes were resolved on 4-15% Bis-Tris SDS gels and transferred to nitrocellulose membranes. Membranes were probed with a mouse anti-human Ubiquitin monoclonal antibody followed by an HRP-conjugated donkey anti-mouse secondary antibody, and then addition of chemiluminescent substrate. Ubiquitin was visualized by measuring ECL. Sample order is the same as in panel A.