Characterization and validation of coatomer as a component in SH4-dependent protein transport. (A) HeLa cells expressing SH4-HASPB-GFP and SH4-YES1-mCherry were transfected with a scrambled siRNA (a–c), siRNAs directed against COPB1 (d–f) and ARCN1 (g–i), and subjected to live-cell confocal microscopy to determine the localization of both SH4 fusion proteins. (B) Equal protein amounts of total cell lysates from transfected and induced cells were subjected to SDS-PAGE and Western blot analysis with antibodies directed against GFP, COPB1, ARCN1, and GAPDH. (C) HeLa cells expressing SH4-HASPB-mCherry were coinjected with recombinant coatomer and an Alexa-488-labeled secondary antibody before transfection with a siRNA directed against COPB1. Thirty hours post-transfection, cells were analyzed by live-cell confocal microscopy to determine the subcellular localization of the SH4 fusion protein in microinjected cells.
