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Fig. 6. IKAP co-localizes with filamin A into membrane ruffles. (A) RT-PCR of IKBKAP, VIP and filamin A. cDNA was prepared from RNA isolated from rat sympathetic neurons (SCG), MEFs and mouse brain. Water was used as a negative control. (B) IKAP association with filamin A. HEK293 cells were transiently transfected with filamin A together with either an empty vector or His-tagged full-length IKBKAP. After 48 hours, cytosolic extracts were prepared and immunoprecipitated with anti-His antibody. Immunoprecipitates were run on SDS-PAGE and analyzed for IKAP and filamin A. Asterisk indicates the 190-kD fragment of filamin A. (C) Pull-down experiment of in-vitro-translated IKAP and filamin A. Unlabeled His-tagged IKAP fragments, S35-labeled (*) N-terminal filamin A fragments and cIKAP-His were in vitro translated with TNT T7 coupled reticulosate lysate system (Promega). Designated in vitro translations were combined and pulled down with anti-His antibody. Upper panel, autoradiogram of pull downs; lower panel, western blot of the IKAP inputs with anti-His antibody. (D) IKAP and filamin A co-immunostaining in IKAP-depleted (m2) and control (scr) MEFs. Human wild-type IKAP and FD-IKAP were expressed in murine IKAP-depleted MEFs as indicated. Cells were plated on coverslips for 24 hours, fixed and stained. (E) Quantification of the percentage of cells with filamin A staining at the membrane ruffles (means ± s.d.; n=3; **P<0.01). (F) Western blot analysis of IKAP and FD-IKAP expression. The quantification (His versus GAPDH; H/G) is representative of two independent transfections. (G) Immunostaining of cells using three different anti-IKAP antibodies (upper panels and lower leftmost panel) and image of the expression of IKAP-GFP in HeLa cells (lower rightmost panel). Boxed images show close-ups of the areas indicated by arrowheads. (H) Western blot analysis of filamin A expression in IKAP-depleted MEFs. (I) Migration of cerebellar granule neurons transiently transfected with designated filamin A (F1) or scr siRNA oligonucleotide was quantified and normalized to control levels (means ± s.d.; n=4; ***P<0.001). (J) Viability of neurons expressing designated filamin A or scr siRNA (means ± s.d.; n=4). Scale bars: 20 µm.
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