Figure 9.
TRIP8b shRNAs are effective and specific for knockdown of TRIP8b expression in transfected HEK293T cells and cultured hippocampal neurons. A, Schematic of Pex5l illustrating the site of targeting by TRIP8b-sh3 (light green, exon 6–7 junction) and TRIP8b-sh4 (dark blue, exon 11). Neither shRNA construct targets Pex5l in the alternatively spliced region. shControl is not predicted to target any known rat or mouse gene. Sense sequences are underlined. The loop sequence is ttcaagaga. TTTTT is the polymerase III transcription termination sequence. B, HEK293T cells were cotransfected with TRIP8b IsoA4 or rescue constructs containing two nucleotide silent mutations in the targeted regions of TRIP8b IsoA4, pCMV-U6 containing either TRIP8b-sh3, TRIP8b-sh4, shControl, or no hairpin, and GFP (to serve as a control for transfection efficiency). After 48 h, cells were lysed, separated by SDS-PAGE, and blotted for TRIP8b, GFP, and α-tubulin, demonstrating that both hairpin constructs are effective for TRIP8b knockdown and are specific for their targeted regions. C, Quantitation of TRIP8b knockdown (n = 3). D, DIV 10 hippocampal neurons were infected with equal titers of FUGWlinker-shControl or FUGWlinker-TRIP8b-sh4 for 11 d, followed by Western blotting for TRIP8b and α-tubulin, confirming that FUGWlinker-TRIP8b-sh4 is effective for knockdown of endogenous TRIP8b in cultured neurons, whereas FUGWlinker-shControl does not alter endogenous TRIP8b compared with uninfected neurons. E, DIV 12 hippocampal neurons were infected with FUGWlinker-shControl or TRIP8b-sh4 and 6 d later stained for GFP (green) and TRIP8b (red), as well as for cell nuclei with Hoechst stain (blue). TRIP8b staining remained present in many neurons infected with FUGWlinker-shControl (top panel, arrows), whereas infection with FUGWlinker-TRIP8b-sh4 reduced endogenous TRIP8b staining in many infected neurons (bottom panel, arrowheads). Camera acquisition settings were identical for top and bottom panels. Scale bar: E, 50 μm.