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Figure 2 | Retrovirology

Figure 2

From: Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function

Figure 2

Immunoprecipitation of HIV-1 RNA from nucleoplasmic fractions. A) Biochemical fractionation for the proteomic analysis. Nuclear extraction scheme showing the various phases of the protocol used to produce the nucleoplasmic fraction. B) Control of nuclear extraction in U2OS cells. The fractions obtained by the protocol outlined in Figure 2A were loaded on a gel for immunoblotting against α-tubulin (upper panel) that shows up only in the cytoplasmic fraction (CF) and against the nuclear protein RecQ (bottom panel) that was present only in the nucleoplasmic fraction (NF). C) Control of HIV-1 RNA associated factor Tat in the NF. Nuclear extracts from U2OS cells (mock), U2OS HIV_Exo_24 × MS2 (exo) or U2OS HIV_Intro_24 × MS2 (intro) were immunoprecipitated for HIV-1 RNA as described above, loaded on SDS-PAGE and blotted against GFP to detect the RNA-bound Tat-CFP protein (IP). Immunoblots for the nuclear extracts against GFP and flag-MS2nls (input) are shown. D) Pulldown of HIV-1 RNA and endogenous MATR3. Whole cell extracts from U2OS cells (mock), U2OS HIV_Exo_24 × MS2 (exo) or U2OS HIV_Intro_24 × MS2 (intro) were immunoprecipitated for HIV-1 RNA as described above, loaded on SDS-PAGE and blotted against MATR3 to detect the RNA-bound endogenous protein (IP). Immunoblots for the whole cell extracts against MATR3 and flag-MS2nls (input) are shown.

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