Figure S2: ZNF268 KRAB Targets GFP Fusion Proteins Into Nucleus in Various Cell Lines

Figure S1

Figure S1(A-C)The expression of various GFP fusion proteins as indicated was detected by western blot with an anti-GFP antibody. (D) KAP1-FLAG was measured with an anti-FLAG antibody. Actin served as loading control.
Figure S2

Figure S2: ZNF268 KRAB targets GFP fusion proteins into nucleus in various cell lines.

GFP control or ZNF268 KRAB-GFP plasmids were transfected into the indicated cells for confocal microscopy analysis.

Figure S3

Figure S3Representative pictures of confocal experiment showed the expression of GFP alone (Control) or various GFP fusion proteins as indicated, the nucleus (DAPI), and their colocalization (Merge).

Figure S4

Figure S4Representative pictures of confocal experiments showed the expression of GFP alone (Control) or various GFP fusion proteins as indicated, the nucleus (DAPI), and their colocalization (Merge). (A) KRABs from different KRAB-ZNFs. (B) KOX1 (C) ZNF300

Figure S5

Figure S5 Interaction of KRAB with nuclear import machinery.

(A) 293T cells were transfected with construct expressing FLAG-tagged Importin . The transfected cells were harvested and cell lysates were prepared for GST pulldown assay with GST protein alone (Control) or GST fusion proteins as indicated. Importin  was detected by western blot with anti-FLAB antibody. The cell lysate used as input control served as positive control. The amount of GST or GST fusion proteins was measured by coomassie blue staining. (B) 293T cells were transfected with construct expressing FLAG-tagged Importin . And GST pulldwon assay was performed as described in A. (C) 293T cells were transfected with constructs expressing FLAG-tagged Importin  (left pannel) or (right pannel) in combination with constructs expressing various KRAB-GFP fusion proteins as indicated. The transfected cells were harvested and cell lysates were prepared for immunoprecipitation by anti-FLAG antibody. The presence of GFP or GFP fusion proteins were detected by western blot with anti-GFP antibody. The amount of Importin  or  protein was measured by western blot with anti-FLAG antibody. Repositioned lanes were from the same blot.

Figure S6

Figure S6: The NLS activity of KRAB is not altered by KAP1 knockdown.

(A) HeLa cells were transfected with control vectoror a vector expressing shRNA specific for KAP1 (shKAP1). Cells were selected with puromycin (2μg/ml) to establish KAP1 knockdown cells. The expression of KAP1 in control or shKAP1 HeLa cells was measured by quantitative RT-PCR. (B) GFP control or ZNF268 KRAB-GFP plasmids were transfected into shKAP1 or sh control HeLa cells. The subcellular distribution of GFP, KAP1 was detected by confocal analysis and merged to DAPI. (C) The histogram of GFP control or ZNF268 KRAB-GFP nucleus localization. Data was presented as the percentage of cells with only nuclear GFP.