Figure 6
RHBDL4-mediated proTGFα-secretion boost is enhanced by transactivation.
(a) Exosome secretion of proTGFα-FLAG in COS7 cells ectopically expressing GRPR is stimulated by PMA or bombesin (bbs), mimicking the effect caused by RHBDL4 overexpression. In absence of BB94 (BB), transactivation by PMA and bbs also affects the extent of pro-peptide cleavage subsequently reducing the amount of secreted pro-peptide (asterisk) in the media fraction. (b) Upon transactivation of COS7 cells with bbs, the secreted 37-kDa form of proTGFα-FLAG was processed to the 6-kDa mature form via a number of intermediates. Of note, due to a low expression the ER-resident 28-kDa form of proTGFα-FLAG was not detected (cf. Fig. 1a,g). (c) Time course of TGFα secretion after PMA stimulation of Hek293T ectopically expressing proTGFα-FLAG was performed in presence of BB94 (BB, 20 μM), DCI (100 μM) or both. (d) PMA and bbs-induced proTGFα-FLAG secretion boost can be suppressed by RHBDL4 knockdown in Hek293 T-REx cells expressing an inducible shRNA but not in control cells. This effect is rescued by ectopically expressed mouse RHBDL4 (mRHBDL4-HA). Cytosolic GFP and ectopically expressed prolactin (Prl) serve as transfection and loading controls.
