Fig. 3: The ECM1a GPR motif determines signaling and tumorigenesis.

a Detection of HA-tagged ECM1a-MT expression in an established cell line by WB analysis. 3D cell growth (b, bars = 400 µm) and quantification (c, data are presented as mean ± SD, n = 3 biologically independent repeats, two-tailed t-test) induced by ECM1a-MT or ECM1a-WT. Xenograft tumor growth (d, data are presented as mean ± SD, n = 10v10 mice, two-tailed t-test) and tissues (e) in ten mice and cellular signaling molecules (f, the actin loading control in f left panel was also used for the same samples in Fig. 4g because these tests were parallelly performed) were inhibited by ECM1a-MT. g Loss of direct binding of integrin αX or β2 to ECM1a-MT, in contrast with the binding to ECM1a-WT, as tested by co-IP. Cellular colocalization of integrin αX or β2 with ECM1a-WT or ECM1a-MT as detected by IF (h, bars = 10 µm) and PLA (i, bars = 10 µm). Detection of integrins αX and β2 and of WT and MT ECM1 by WB analysis of cell membrane extracts (j) and detection of integrins αX and β2 (k) by co-IP/WB analysis after cell surface labeling with biotinylation reagents.