Impaired Caveolae Function and Upregulation of Alternative Endocytic Pathways Induced by Experimental Modulation of Intersectin-1s Expression in Mouse Lung Endothelium
Figure 1
Experimental modulation of ITSN-1 expression in mouse lungs. (a) Myc-SH3A domain of ITSN-1s is efficiently expressed in mouse lungs, at 48 h alter delivery of the plasmid cDNA/liposome complexes. Mouse lung lysates (70 μg total protein/lane) of control and myc-SH3A-transduced mice were subjected to 4–20% SDS PAGE, transferred to nitrocellulose membrane and immunoblotted with myc Ab. (b) siRNA-mediated knockdown of ITSN-1s in mouse lungs. Lung lysates (70 μg total protein/lane) of controls and mice injected with liposomes/ITSN-1s siRNA complexes were resolved on 4–20% SDS PAGE and the electrophoretograms were transferred to nitrocellulose membrane followed by Western blot with Abs against ITSN-1, actin (as a loading control) and cav-1. Note the efficient knockdown of ITSN-1s protein for 96 h, starting at day 3 (3 d) alter siRNA delivery. (c) Densitometric analysis of representative HyBlot CL films demonstrates that treatment with ITSN-1s siRNA/liposome complexes downregulates efficiently ITSN-1s protein in mouse lungs. (d) Quantitative RT PCR of ITSN-1s mRNA in ITSN-1s siRNA treated mouse lungs. Values were normalized to the house-keeping gene cyclophilin and reported relative to control lungs (set at 1). (e) Chronic inhibition of ITSN-1s expression for 21 consecutive days by repeated delivery of ITSN-1s siRNA/liposomes complexes. Lung lysates of control and ITSN-1s-siRNA-treated mice were analyzed by Western blot with Abs against ITSN-1s and actin, as loading control, at several time points alter delivery, as indicated. (f) Conventional RT-PCR of ITSNs isoforms expressed in mouse lungs. RT PCR reactions were amplified as described under Materials and Methods using appropriate primer sets for the target genes, ITSN-1s, ITSN-2s and ITSN-2L, and a house-keeping gene cyclophilin. 5-μL of PCR products were subjected to 1.2% agarose gel electrophoresis and visualized using an Alpha Innotech AlphaImager II system. All data shown are representative for 3 different experiments, (3 mice/experimental condition).
