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. 2017 Jul 17;8(38):64050–64065. doi: 10.18632/oncotarget.19300

Figure 1. Characterization of macrophages stably expressing biotinylated Sp110.

Figure 1

(A) The structure of plasmids used for constructing the stably-transfected RAW264.7 cells. (B) Verification of biotinylated Sp110. Western blot analysis of the nuclear lysates from the RAW264.7 cells stably transfected with the indicated plasmids. Wild-type RAW264.7 cells were used as a control. (C) Bioinformatic prediction and experimental validation of Sp110 nuclear localization. Top: Predicted nuclear localization signal of Sp110 protein. Bottom: Immunofluorescence staining of RAW264.7, RAW-BAP-Sp110, and RAW-BAP-Sp110-P2A-BirA cells with an anti-Flag antibody. Nuclei were stained with DAPI. Scale bar = 5 μm. (D) Validation of the interaction between biotinylated Sp110 and Hspa8. IP of the nuclear lysates prepared from RAW-BAP-Sp110-P2A-BirA or RAW-BAP-P2A-BirA cells using streptavidin-conjugated agarose resin. The presence of Sp110 and Hspa8 was detected by immunoblotting. (E) Flow cytometric analysis of the apoptosis of RAW264.7, RAW-BAP-P2A-BirA, RAW-BAP-Sp110, and RAW-BAP-Sp110-P2A-BirA cells. The cells were infected with H37Ra at an MOI of 5 for 24 h. Uninfected cells were used as controls.