JCS02472 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 - Fig. S1. HeLa cells were fixed as described in Fig. 1A: first column: avoid 1°Ab; second column: avoid 2° Ab; 3^rd column: 2 mg/ml rabbit IgG; 4^th column: 2 mg/ml affinity-purified anti-rp3 antibody. Specific nuclear labeling derived from the affinity-purified anti-rp3 antibody is not observed in the HeLa cells labeled in any other control conditions.

• Supplemental Figure 2 - Fig. S2. Binary two-hybrid analyses were carried out by using the pPC97 (bait)/pPC86 (prey) system. The figure shows the X-gal filter assay results of Y190 yeast cells co-transformed with (A) rp3 (in pPC97) and pPC86 empty vector, (B) rp3 (in pPC97) and SATB1 (in pPC86), (C) Tctex-1 (in pPC97) and SATB1 (in pPC86), and (D) pPC97 empty vector and SATB1 (in pPC86). Only yeasts co-express rp3 and SATB1, but not other controls, exhibited blue colonies.

• Supplemental Figure 3 - Fig. S3. RT-PCR analysis of rp3, Tctex-1, SATB1 in HeLa and HEK cells. PCR reactions were carried out by using RNA isolated from HeLa and HEK cells using primers for rp3 (5’-cggaattcgagccggcgctaccatggaggag-3’ and 5’-gcatctagactcgaggtcagttaaagaacaatagc-3’), Tctex-1 (5’-tgaattcatggctggtggttagagg-3’ and 5’-agtgaaccagtggaccac-3’), and SATB1 (5’-gctctagagtaaataccagtggcactgttgaacg-3’ and 5’-ccaacaatacttgaaccaccctccc-3’)

• Supplemental Figure 4 - Fig. S4. Nuclear extracts (60 mg) obtained from Jurkat T cells and HEK cells were separated in a SDS-PAGE and immunoblotted with anti-SATB1 mAb. The appearance of single bands of ~86 kDa in both samples not only suggested that SATB1 is expressed in these cells, but also confirms the Ab specificity.

• Supplemental Figure 5 - Fig. S5. CEBP/b was unable to translocate rp3 into nucleus. Confocal images of HEK cells double-transfected with CEBP/b and Flag-rp3 and immunolabeled with anti-CEBP/b (red) and anti-Flag Abs (green). DAPI (blue): nuclear labeling. Bar= 10 mm.

• Supplemental Figure 6 - Fig. S6. Endogenous rp3 and SATB1 interacted each other. Cytosolic and nuclear extracts from adult rat cerebral cortex were immunoprecipitated with anti-SATB1 Ab and immunoblotted for either SATB1 or rp3. The bottom panel showed the direct immunoblotting of SATB1.

• Supplemental Figure 7 - Fig. S7. To estimate the proportion of dynein-free rp3 that binds SATB1, we first removed IC from nuclear extracts of either rat brain or HEK cells by anti-IC antibody. The post-IP sup was then subjected to a second-round IP using anti-SATB1 antibody. The SATB1 immunoprecipitates as well as progressive amount of post-IP sups were loaded side-by-side on SDS-PAGE, followed by immunoblotting with either anti-SATB1 or anti-rp3 antibodies using the ECL method (diagramed in A). (B) The nuclear extracts pre-absorbed with IC-antibody lack IC signals on immunoblotting, demonstrating the IC depletion is completed. rp3 was co-immunoprecipiated with SATB1 in the IC-depleted nuclear sup (C, lane 1), reiterating that the rp3 associated with SATB1 is outside the IC and dynein complex. The amount of rp3 pulled down by SATB1 is rather abundant, in fact, it appears to be more abundant than the matched total post-IC-IP nuclear sup (compared lane1 vs. lane 7). The highly abundant rp3 in the SATB1 IP suggests that a large proportion, and perhaps the majority, of free-pool of rp3 binds to SATB1 in nucleus.

• Supplemental Figure 8 - Fig. S8. Specificity of complex I and II generated by SATB1. ³²P-labeled and unlabeled (competitor) Bcl-2 MAR oligos were mixed in different ratios as indicated, and incubated with nuclear extracts of HEK cells transfected with SATB1. Complexes I and II were specifically diminished by excess competitors. The bands near the bottom (arrowhead) of the figure represent non-specific retarded bands which cannot be removed by the unlableled competitor.

• Supplemental Figure 9 - Fig. S9. Immunodepletion of dynein IC. Nuclear extract from SATB1, Flag-rp3 double-transfected HEK cells was IPed by anti-IC Ab to remove the endogenous IC. 30 mg nuclear extracts prior to the IP (Pre-IP) and after the IP (DIC) were analyzed by immunoblotting to demonstrate that IC was completely removed by the IP procedures. Whereas, the nuclear levels of SATB1 and rp3 were largely unaltered.