Fig. 5

SDF-1α-stimulated ERK1/2 phosphorylation was mediated by endogenous CXCR4. a RT-PCR. RNA isolated from HEK293 cells was subjected to RT-PCR using gene-specific primer sets. The PCR products were separated using 2% agarose gels (SM: 1 kb ladder size marker). b HEK293 cells and HeLa cells lacking CXCR4 (CXCR4-KO) or CXCR7 genes (CXCR7-KO) were established by CRISPR/Cas9 gene-deletion methods. CXCR4-deficient HEK293 cells were transfected with CXCR7 plasmids (CXCR4-KO/CXCR7). Cells were treated with SDF-1α for 10 min and harvested. Cell lysates were used for western blot analysis with anti-pERK or ERK antibodies. c Time-dependent ERK phosphorylation by SDF-1α in wild-type HEK293 cells (wild), CXCR4- or CXCR7-deficient HEK293 cells (CXCR4-KO or CXCR7-KO). d The efficiency of SDF-1α inhibition on isoproterenol-stimulated cAMP generation in CXCR4- or CXCR7-deficient HEK293 cells. The cells were transfected with receptor gene plasmids and pGlo22F containing a cAMP detector gene plasmid. Cells were treated with SDF-1α for 10 min prior to isoproterenol (Iso) or no treatment (Veh.). Real-time intracellular cAMP production was measured as luminescence. Results are the average of three independent experiments.