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Fig. 5 | Cell & Bioscience

Fig. 5

From: Analysis of CCR2 splice variant expression patterns and functional properties

Fig. 5

Analysis of the cellular responses mediated by CCR2A or CCR2B. A Specific interaction between CCR2 variants and Gα isotypes. HEK293 cells expressing C-terminal SmBiT-tagged receptors and N-terminal LgBiT-tagged mini-G proteins were treated with MCP-1 and luciferase activity was measured by a luminometer. B ERK phosphorylation. Wild type cells and CCR2 KO cells were treated with MCP-1 for 10 min (left upper panel). CCR2 KO cells and CCR2 variant-reconstituted cells were treated with MCP-1 for 10 min (left lower panel). All cells described in the figure were treated with MCP-1 for different time (right panel). Cell extracts were subjected to western blotting with anti-phospho-extracellular signal-regulated kinase (pERK) antibodies or anti-ERK antibodies. C, D Intracellular Ca2+ measurements assessed using the enzyme complementation assay. **p > 0.01 (C, D) HEK293 cells (C) or HEK293-Gqi cells (D) expressing LgBiT-MYLK2s (described as M2S) and calmodulin-SmBiT (described as CaM) together with CCR2A or CCR2B were treated with MCP-1, and luciferase activity was determined using a luminometer. The graph on the right side shows the maximum fold change in luciferase activity induced by MCP-1. ##p > 0.01, in comparison between CCR2A and CCR2B. V: vector (E) MCP-1 dose dependency of the calcium responses. HEK293-Gqi cells expressing the calcium probes with receptors were treated with different doses of MCP-1. The graph shows the maximum fold change in luciferase activity. The numbers below each curve designate EC50 value (M) of MCP-1. F Reporter gene assay. Cells containing the Gqi construct were transfected with the serum response element-luciferase (SRE-Luc) gene and CCR2 variants. After overnight starvation, the cells were treated with MCP-1 for 6 h and lysed in lysis buffer. The luciferase activity of each extract was measured using a luminometer. NT: non-treated. ##p > 0.01, difference between CCR2A and CCR2B. **p > 0.01, compared with non-treated. All experiments were conducted at least three independent times

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