Effect of PMA and the C-terminal domain of Flt1 on regulating the abundance and cleavage of Flt1.

<p><b>Panel A and B:</b> HEK293 cells transfected with epitope tagged Flt1 and incubated with PMA (30 nM) and/or GF109 (1 µM) and for 24 hrs. Lysates were immunoblotted with Flag and HA and with HSP90 as a loading control. PMA increases the abundance of full length (FL) Flt1 and increases the cleaved N-terminal fragment in conditioned media (condt. media) and the corresponding C-terminal fragment in HEK293 cell lysates. The effect of PMA is blocked by the PKC inhibitor GF109293X (GF109) indicating that the PMA effect is mediated via activation of PKC. Representative immunoblot in A and quantitative pooled data from 3 experiments are shown in B. *p<0.05, **p<0.001. <b>Panel C and D:</b> HEK293 cells transfected with Flt1 or Flt1 ΔCTD and incubated with PMA and for 24 hrs. PMA has a greater effect on increasing Flt1 ΔCTD abundance and on increasing N-terminal cleavage. Representative immunoblot in C and quantitative pooled data of the N-term fragment in condt. Media from 4 experiments are shown in D. *p<0.05 by Mann Whitney Rank sum test. Small arrowheads in Panel C indicate the position of full length FLT1 (FL FLT1) and of the C-terminal deleted form of Flt1 (Flt1 ΔCTD). <b>Panel E:</b> HEK293 cells transfected with Flt1 ΔCTD and incubated with PMA (30 nM) and/or GM6001 (1 µM) for 24 hrs. Conditioned media (condt. media) was blotted with HA while lysates were blotted with tubulin. PMA increases while GM6001 reduces the abundance the N-terminal HA tagged Flt1 fragment in CM.