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1.
FIG. 1.

FIG. 1. From: PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases.

PIAS proteins interact with SUMO-1 and Ubc9 in vitro. ARIP3, ARIP3Δ347-418, ARIP3Δ467-487, Miz1, PIAS1, and PIAS3 were labeled with [35S]methionine by translation in vitro and incubated with glutathione-Sepharose-bound GST, GST-SUMO-1, or GST-Ubc9. After extensive washings, bound proteins were eluted with SDS sample buffer, resolved by SDS-10% PAGE, and visualized by fluorography. Lanes i, input samples representing 10% of the amount of labeled proteins incubated with the matrices.

Noora Kotaja, et al. Mol Cell Biol. 2002 July;22(14):5222-5234.
2.
FIG. 9.

FIG. 9. From: PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases.

ARIP3 enhances the sumoylation of AR and c-Jun in vitro. (A) Effect of ARIP3 on sumoylation of in vitro-translated AR. 35S-labeled, in vitro-translated AR was incubated with purified GST-SUMOGG-1 (in all reactions), GST-SAE1 plus GST-SAE2 (in all reactions), and GST-Ubc9 in the presence or absence of GST, GST-ARIP3, or GST-ARIP3Δ347-418 as indicated. Samples were resolved by SDS-PAGE and subjected to fluorography. (B) ARIP3 enhances the sumoylation of purified recombinant c-Jun. His-tagged c-Jun was incubated with GST-SUMOGG-1, GST-SAE1 plus GST-SAE2, and GST-Ubc9 in the presence of GST alone, GST-ARIP3, GST-ARIP3Δ347-418, GST-ARIP3(W383A), or GST-ARIP3Δ467-487 as indicated. Samples were resolved by SDS-PAGE and immunoblotted with the anti-His antibody. Arrowheads indicate sumoylated forms of proteins.

Noora Kotaja, et al. Mol Cell Biol. 2002 July;22(14):5222-5234.
3.
FIG. 3.

FIG. 3. From: PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases.

PIAS proteins and SUMO-1 colocalize in nuclear granules. COS-1 cells were cotransfected with expression plasmids encoding EGFP-SUMO-1 and FLAG-ARIP3, FLAG-ARIP3Δ347-418, FLAG-ARIP3Δ467-487, FLAG-PIAS1, FLAG-PIAS3, or FLAG-Miz1. Immunofluorescence labeling was performed with the M2 antibody against the FLAG epitope and a rhodamine-conjugated secondary antibody, and cells were analyzed as described for Fig. 2. Images were collected separately (EGFP was excited at 488 nm, and lissamine-rhodamine-conjugated anti-mouse IgG for M2 was excited at 568 nm) and merged as depicted.

Noora Kotaja, et al. Mol Cell Biol. 2002 July;22(14):5222-5234.
4.
FIG. 8.

FIG. 8. From: PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases.

ARIP3 and PIAS1 enhance the sumoylation of AR in HeLa cells. HeLa cells seeded onto six-well plates were cotransfected with 100 ng of pcDNA-Flag-hAR; 100 ng of pFLAG-CMV2, pFLAG-ARIP3, or pFLAG-PIAS1; and 0, 10, or 50 ng of pSG5-His-SUMO-1. Cells were lysed in RIPA-1 buffer, and the lysates were immunoblotted with the anti-AR antibody. The difference between the migration of AR forms sumoylated with endogenous SUMO-1 and that of ectopically expressed SUMO-1 is due to the His tag present in the SUMO-1 expression vector. The identities of sumoylated AR forms were verified by immunoblotting the anti-AR antibody-immunoprecipitated AR with the anti-SUMO-1 antibody (not shown).

Noora Kotaja, et al. Mol Cell Biol. 2002 July;22(14):5222-5234.
5.
FIG. 6.

FIG. 6. From: PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases.

Zinc finger region of ARIP3 is required for the recruitment of other sumoylated proteins. (A) FLAG-tagged wild-type ARIP3, ARIP3Δ347-418, ARIP3Δ467-487, and ARIP3Δ346-475 were coexpressed in COS-1 cells without or with pSG5-His-SUMO-1 as described for Fig. 4. Cells were lysed in RIPA-1 buffer, and samples from the lysate were immunoblotted with the anti-FLAG antibody. (B) Expression vectors encoding EGFP, EGFP-ARIP3 (341-418), or EGFP-ARIP3 (341-490) were cotransfected into COS-1 cells without or with pSG5-His-SUMO-1. Cells were lysed in RIPA-1 buffer, and lysates were immunoblotted with an antibody against GFP. (C) The rest of the lysates from panel A were immunoprecipitated (IP) with the anti-FLAG antibody, and immunoprecipitates were immunoblotted with the anti-SUMO-1 antibody. (D) The rest of the lysates from panel B were immunoprecipitated with the anti-GFP antibody, and immunoprecipitates were immunoblotted with the anti-SUMO-1 antibody. WB, Western blotting.

Noora Kotaja, et al. Mol Cell Biol. 2002 July;22(14):5222-5234.
6.
FIG. 2.

FIG. 2. From: PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases.

Localization of PIAS proteins and SUMO-1 in COS-1 cells. Cells were transfected with expression plasmids encoding FLAG-ARIP3, FLAG-ARIP3Δ347-418, FLAG-ARIP3Δ467-487, FLAG-PIAS1, FLAG-PIAS3, FLAG-Miz1, or EGFP-SUMO-1 as described in Materials and Methods. Immunofluorescence labeling was performed with the M2 antibody against the FLAG epitope and a rhodamine red X-conjugated secondary antibody (A to F). Cells were analyzed by using a Bio-Rad MRC-1024 confocal laser scanning system connected to a Zeiss Axiovert 135 M microscope. EGFP was excited at 488 nm, and rhodamine red X was excited at 568 nm.

Noora Kotaja, et al. Mol Cell Biol. 2002 July;22(14):5222-5234.
7.
FIG. 10.

FIG. 10. From: PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases.

PIAS proteins enhance the sumoylation of GRIP1. COS-1 cells grown on 6-cm-diameter plates were transfected with 0.7 μg of empty pFLAG-CMV2 vector or a vector encoding the FLAG-tagged PIAS protein, 0.9 μg of pSG5 or pSG5-GRIP1, and 0.9 μg of pSG5 or pSG5-His-SUMO-1. The cells were harvested 48 h after transfection and lysed in RIPA-2 buffer containing 10 mM NEM. Five percent of the lysate was immunoblotted with the anti-GRIP1 antibody (A), and the rest was subjected to immunoprecipitation (IP) with the anti-FLAG antibody followed by immunoblotting with the anti-GRIP1 antibody (B). WB, Western blotting. (C) The amounts of PIAS proteins were verified by immunoblotting the cell lysates with the anti-FLAG antibody. (D) The predicted zinc-binding region of PIAS1 is required for stimulation of the sumoylation of GRIP1. COS-1 cells were cotransfected with 0.9 μg of pSG5-GRIP1; 0.7 μg of pFLAG-CMV2, pFLAG-PIAS1, pFLAG-PIAS1(W372A), or pFLAG-PIAS1Δ310-407; and 0.9 μg of pSG5 or pSG5-His-SUMO-1. Cells were lysed in RIPA-2 buffer, and lysates were immunoblotted with either the anti-GRIP1 antibody or the anti-FLAG antibody.

Noora Kotaja, et al. Mol Cell Biol. 2002 July;22(14):5222-5234.
8.
FIG. 5.

FIG. 5. From: PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases.

ARIP3 interacts with other sumoylated proteins. COS-1 cells were transfected with 1 μg of empty pFLAG-CMV2 or pFLAG-ARIP3 and 1.5 μg of empty pSG5 or pSG5-His-SUMO-1. Forty-eight hours after transfection, cells were collected and lysed either in RIPA-1 buffer containing 10 mM NEM or in a denaturing buffer containing 2% SDS in 10 mM Tris-HCl (pH 8.0)-150 mM NaCl. Samples lysed in the latter buffer were heated at 95°C for 10 min. Before immunoprecipitation (IP), these samples were diluted (1:10) with a mixture of 10 mM Tris-HCl (pH 8.0) and 150 mM NaCl containing 1% Triton X-100. Five percent of the cell extracts were immunoblotted with the anti-FLAG antibody (A), and the rest of the samples were immunoprecipitated with the anti-FLAG antibody followed by immunoblotting with the anti-SUMO-1 antibody (B) or the anti-FLAG antibody (C). WB, Western blotting.

Noora Kotaja, et al. Mol Cell Biol. 2002 July;22(14):5222-5234.
9.
FIG. 4.

FIG. 4. From: PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases.

Modification of ARIP3 and other PIAS proteins by SUMO-1 in mammalian cells and in vitro. (A) COS-1 cells grown on 6-cm-diameter dishes were transfected with 1 μg of pFLAG-ARIP3, pFLAG-Miz1, pFLAG-PIAS1, or pFLAG-PIAS3 and 1.5 μg of empty pSG5 vector or pSG5-His-SUMO-1. Forty-eight hours after transfection, the cells were lysed in RIPA-1 buffer containing 10 mM NEM, and the cell lysates were immunoblotted with the M2 FLAG antibody. (B) Sumoylation of PIAS proteins in vitro. In vitro-translated, 35S-labeled PIAS proteins were incubated with purified GST-SUMOGG-1 in the presence (+) or absence (−) of GST-SAE1 plus GST-SAE2 and GST-Ubc9 as indicated. Reactions were stopped by adding SDS-PAGE sample buffer, and the samples were resolved by SDS-PAGE and subjected to fluorography. (C) Sumoylation of purified GST-ARIP3, GST-ARIP3Δ347-418, and GST-ARIP3Δ467-487 (1 μg each) under conditions described for panel B. The reaction mixtures were immunoblotted with an anti-ARIP3 antibody.

Noora Kotaja, et al. Mol Cell Biol. 2002 July;22(14):5222-5234.
10.
FIG. 7.

FIG. 7. From: PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases.

Role of ARIP3 domains in the interaction with AR and effect of ARIP3 on protein sumoylation. (A) COS-1 cells grown on 6-cm-diameter plates were transfected with 0.7 μg of empty pFLAG-CMV2 vector or pFLAG-ARIP3, 0.9 μg of pSG5 or pSG5-rAR, and 0.9 μg of pSG5 or pSG5-His-SUMO-1. The cells were supplied with 100 nM testosterone 24 h after transfection and were collected 48 h after transfection and lysed in RIPA-2 buffer in the presence (+) or absence (−) of NEM. Five percent of the lysate was immunoblotted with the K333 antibody against AR, and the rest of the sample was subjected to immunoprecipitation (IP) with the anti-FLAG antibody. Immunoprecipitates were blotted with anti-AR antibody. WB, Western blotting. (B) The zinc finger region of ARIP3 is required for the NEM-dependent interaction with AR. Versions of FLAG-tagged ARIP3 with different deletions were coexpressed with AR in COS-1 cells. Cells were lysed in RIPA-2 buffer containing 10 mM NEM, and immunoblotting of cell extracts and immunoprecipitation were performed as described for panel A. Arrowheads indicate sumoylated forms of AR. (C) Overexpression of ARIP3 enhances the sumoylation of endogenous COS-1 cell proteins. ARIP3, ARIP3Δ347-418, or ARIP3Δ467-487 were coexpressed in COS-1 cells without or with SUMO-1. Cells were lysed in RIPA-1 buffer, and lysates were immunoblotted with the anti-SUMO-1 antibody. wt, wild type.

Noora Kotaja, et al. Mol Cell Biol. 2002 July;22(14):5222-5234.
11.
FIG. 11.

FIG. 11. From: PIAS Proteins Modulate Transcription Factors by Functioning as SUMO-1 Ligases.

The SUMO-1-tethering region and the ligase domain are critical for the ability of PIAS proteins to coregulate AR-dependent transcription. HeLa (A) and COS-1 cells (B) cultured on 12-well plates were cotransfected with 200 ng of pARE2TATA-LUC, 20 ng of pCMVβ, 5 ng of pcDNA-Flag-hAR, and 10, 50, or 100 ng of pFLAG-ARIP3, pFLAG-ARIP3 (W383A), or pFLAG-ARIP3Δ467-487 and treated or not treated with 100 nM testosterone (T). After normalization for transfection efficiency by using β-galactosidase activity, reporter gene activities were expressed relative to those of AR plus T without a coregulator (set at 100). wt, wild type. (C) The same experiment as in panel B, but instead of ARIP3 expression plasmids, COS-1 cells were cotransfected with pFLAG-PIAS1, pFLAG-PIAS1(W372A), or pFLAG-PIAS1Δ310-407. (D and E) Effect of cotransfected ARIP3 on the AR mutant with the SUMO-1 attachment lysines mutated to arginines [AR(K→R)] compared to that on wild-type AR. The experimental conditions were the same as those in panels A and B. The values represent means ± standard deviations from three to six independent experiments.

Noora Kotaja, et al. Mol Cell Biol. 2002 July;22(14):5222-5234.

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