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. Author manuscript; available in PMC: 2013 Feb 1.
Published in final edited form as: Lab Invest. 2012 May 28;92(8):1213–1233. doi: 10.1038/labinvest.2012.80

Figure 5. CCL2 promotes disassembly of the AJ.

Figure 5

(A) Confluent hCMEC/D3 were treated with 200 ng/mL CCL2 or diluent. Cells were lysed and 140 µg of total protein were immunoprecipitated and evaluated by Western blot. Optical density of pulled-down protein was compared to OD of immunoprecipitating protein. Protein association in response to CCL2 was compared to control cells at the same time point and is reported as fold change. Findings are consistent when VE-cadherin, β-catenin, or α-actinin is used to pull-down other components of the AJ. Blots are representative of 3 independent experiments. An intervening time point was removed from the image and is represented by a dividing line in the blot. C = control lysate. Association of VE-cadherin with β-catenin and α-actinin is reduced after 30 minutes of CCL2 treatment, indicating loss of AJ integrity. Adherens junction disassembly corresponds to VE-cadherin and β-catenin phosphorylation. (C–F) Cytoplasmic, membrane, nuclear, and cytoskeletal fractions were isolated using different buffers from CCL2- or vehicle-treated primary HBMVEC. 10 µg of total protein from each fraction was evaluated by Western blot. VE-cadherin was quantifiable in only the membrane and cytoskeletal fractions. Optical density of VE-cadherin and β-catenin were compared to OD of loading control specific to each fraction. Changes in protein localization in response to CCL2 were compared to control cells at the same time point and are reported as fold change. n=3. (*) p < 0.05, (#) p < 0.01. (C–D) After 30 minutes of CCL2 treatment, association of VE-cadherin with the cytoskeleton is reduced. This corresponds with VE-cadherin phosphorylation and decreased association with β-catenin and α-actinin. (E–F) In response to 30 minutes of CCL2 treatment, β-catenin localization to the cytoplasm, nucleus, and cytoskeleton is reduced, while its localization in the membrane is increased. This corresponds with β-catenin phosphorylation and AJ disassembly and indicates that β-catenin is being sequestered at the membrane away from other cellular compartments in response to 30 minutes of CCL2 treatment.