Figure 3. Catalytic activity of RNase is not required for activation of and binding to EGFR.

(A) IB of secreted proteins from conditioned media (CM) in HeLa cells transiently transfected with the indicated plasmids. Signals were quantified using ImageJ. Relative density of Flag-bRNaseA-WT was set as 1.

(B) Detection of RNase enzyme activity in CM-secreted proteins collected from (A). Following the addition of fluorescent substrate, the mixtures were subjected to RNaseAlert^® Lab Test kit. Signals were monitored by a BioTek Synergy™ Neo real-time fluorometer.

(C) HeLa cells were treated with secreted proteins from CM as described in (A) for 5 min and subjected to IB with the indicated antibodies.

(D) HeLa cells were treated with CM-secreted proteins as described in (A) for 15 min, fixed and stained with EGFR and bRNaseA antibodies, and subjected to Duolink in situ PLA. Bar, 10 μm. Right, the number of interactions per cell normalized to the amount of the secreted proteins.

(E) IB of CM-secreted proteins in HeLa stable transfectants as indicated. Signals were quantified using ImageJ. Relative density of HeLa-Flag-bRNaseA-WT was set as 1.

(F) Detection of RNase enzyme activity in CM collected from (E).

(G) HeLa cells were treated with CM-secreted proteins as described in (E) for 15 min, fixed and stained with EGFR and Flag antibodies, and subjected to Duolink in situ PLA. Bar, 10 μm. Right, the number of interactions per cell normalized to the amount of the secreted proteins.

(H) Cell morphological changes of HeLa cells treated with the indicated proteins for 3 days. Bar, 50 μm.

(I) IB of HeLa cells lysates extracted from (H).

(J) IB of HeLa cells treated with the indicated proteins or water (−) for 5 min.

(K) IB of CM-secreted proteins in HeLa stable transfectants expressing the indicated plasmids. Signals were quantified using ImageJ. Relative density of HeLa-Flag-ANG-WT was set as 1.

(L) HeLa cells were treated with CM-secreted proteins as indicated in (K) for 5 min and subjected to IB with the indicated antibodies. These experiments were performed in duplicate.

(M) HeLa cells were treated with CM-secreted proteins as indicated in (K) for 15 min, fixed and stained with EGFR and Flag antibodies and subjected to Duolink in situ PLA. Bar, 10 μm. Bottom, the number of interactions per cell normalized to the amount of the secreted proteins.

All error bars represent mean ± SD. *p < 0.05, ***p < 0.001, Student’s t-test. NS, not significant. Data are representative of 3 independent experiments, randomly chosen in 3 different fields.

See also Figure S3.