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. 2019 Jan 4;10(2):133–151. doi: 10.18632/oncotarget.26512

Figure 7. Subcellular localization and interaction of AnxA6 with RasGRF2.

Figure 7

(A–C) The indicated cell lines were grown to 70% confluency, treated with or without EGF, harvested by scrapping in ice-cold PBS and fractionated by differential velocity centrifugation as described in methods. The post-nuclear supernatants (A) or the various fractions from control and AnxA6 up-regulated MDA-MB-468 cells (B) and control and AnxA6 down-regulated MDA-MB-468 cells (C) were analyzed by western blotting. (D–E) Detection of AnxA6, RasGRF2, Ras proteins and β-actin in GST-Raf-1 binding domain (RBD) pull downs and co-IPs. Equal amounts of whole cell extracts prepared as described in Figure 4A and B from control and AnxA6 overexpressing MDA-MB-468 cells, were used in Ras-GTP pull down assays (RBD) or immunoprecipitation using antibodies to pan-Ras (IP: Ras) or AnxA6 (IP: AnxA6). GST-RBD bound proteins as well as protein A/G bound immune complexes were analyzed by western blotting using antibodies against Ras, RasGRF2, AnxA6 and β-actin. Blots were revealed by ECL. Shown are shorter (D) and longer (E) exposures. EV: empty vector transfected; A6: flag-AnxA6 transfected; NSC: no silencing control transfected; and A6sh: shRNA targeting AnxA6 transfected MDA-MB-468 cells. PNS: Post-nuclear supernatant; H-Mem: Heavy membranes; L-Mem: lighter membranes; Cyt: cytosol.