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. 2003 Nov;23(21):7498–7509. doi: 10.1128/MCB.23.21.7498-7509.2003

FIG. 7.

FIG. 7.

The subcellular localization of Sp110b was altered from the nucleus to an area around the cytoplasmic surface of ER membranes by core expression. (A) Indirect immunofluorescence analysis was performed on COS-7 cells transfected with pCMV-core (panel 1), pCA-Sp110 (panel 2), pCA-Sp110 with pSG5-RARα (panels 3 to 5), pCA-Sp110 with pCMV-core (panels 6 to 8), pCMV-Sp110b (panel 9), pCMV-Sp110b with pSG5-RARα (panels 10 to 12), pCMV-Sp110b with pCMV-core (panels 13 to 15), pCMV-core(6162 M) (panel 16), pCMV-Sp110b with pCMV-core(6162 M) (panels 17 to 19), pCMV-Sp110b(1-276 + 454-539) (panel 20), pCMV-Sp110b(1-276 + 454-539) with pCMV-core (panels 21 to 23), or pCMV-Sp110b and pCMV-core with pcDNA-Sp110b(389-453) (myc-CBR fragment) (panels 24 to 29) and on HeLa cells transfected with pKS+/CMV (panel 30) or pCMV-core (panels 31 to 33) following treatment with 300 U of gamma interferon/ml to allow the detection of endogenous Sp110b. The primary antibodies used were anti-FLAG (panels 2, 3, 6, 9, 10, 13, 17, 20, 21, and 27) (green), anti-Myc (panel 24) (green), anti-Sp110(b) (panels 30 and 31) (green), anti-core (panels 1, 7, 14, 16, 18, 22, 25, 28, and 32) (red), and anti-RARα (panels 4 and 11) (red). Merged images of green and red signals are shown in panels 5, 8, 12, 15, 19, 23, 26, 29, and 33. DAPI was used to visualize nuclear staining (right panels). (B) Subcellular fractionation was performed on cells transfected with 1 μg of pCMV-Sp110b (lanes 1 to 3), 1 μg of pCMV-core (lanes 4 to 6), and 1 μg each of pCMV-Sp110b and pCMV-core (lanes 7 to 9). The transfectants were homogenized and separated into nuclear (N), microsomal-membrane (MM), and cytosolic (C) fractions by centrifugation as described in Materials and Methods. The FLAG-tagged Sp110b, core, α-tubulin, and SC-35 proteins in those fractions were detected by immunoblot analysis.