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. 2018 Dec 18;294(6):2046–2059. doi: 10.1074/jbc.RA118.004630

Figure 3.

Figure 3.

INSIG1 promoted the degradation of HIV-1 Gag. A, INSIG1 had no effect on LTR activity. 293T cells were transfected with LTR-luc (LTR-promoted luciferase expression plasmid), Tat, and β-gal and with or without INSIG1. The measured luciferase activity was normalized by a β-gal assay. Shown is mean ± S.D. (error bars) from three independent experiments. B, expression of ES plasmid (NLENY1-ES-IRES), Env, or VSV-G plasmids, respectively, in the presence of INSIG1 in 293T. The expression of relevant proteins was detected by Western blotting. INSIG1 decreased the protein level of Gag encoded from NLENY1-ES-IRES plasmid but does not change the level of Env and VSV-G. C, pCMV-promoted Gag-pol was expressed in the presence of INSIG1, INSIG1-D205A, INSIG1-KH mut, or INSIG1-ΔN mut in 293T cells. Pr55Gag levels in the transfected cells were detected by Western blotting. WT and three mutant INSIG1s all reduced the protein levels of HIV-1 Gag in 293T cells. D, HA-tagged INSIG1 or INSIG1-D205A was expressed with FLAG-tagged SCAP in 293T cells. 36 h after transfection, the cultures were treated with 5 μg/ml cholesterol for 5 h and harvested, lysed, and immunoprecipitated (IP) with anti-HA mAb to precipitate INSIG1. INSIG1 and SCAP in pellets and supernatants were detected by Western blotting with anti-HA or anti-FLAG antibodies. SCAP was co-immunoprecipitated with WT but not D205A mutant INSIG1.