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. 2014 Dec 22;35(2):344–355. doi: 10.1128/MCB.00926-14

FIG 4.

FIG 4

DGCR14 was associated with BAZ1B and RSK2. (A) Western blotting of DGCR14 in 68-41 cells. For biochemical purification, nuclear extracts of DGCR14-Flag-expressing 68-41 cells were prepared. After fractionation by glycerol density gradient centrifugation, each fraction was analyzed by Western blotting with the antibodies indicated. Fractions (Fr. No.) 2 and 3 were subjected to further purification with anti-Flag M1 agarose affinity gel. (B) Biochemical purification of DGCR14-associated proteins. After the establishment of DGCR14-Flag-expressing 68-41 cells, nuclear extracts were prepared and purified with anti-Flag M1 agarose affinity gel. Samples were eluted by Flag peptide and subjected to SDS-PAGE and silver stained, and proteins were identified by MALDI-TOF MS (see Table S2 in the supplemental material). (C) Immunoprecipitation assays of DGCR14 and RSK2 in 293T cells. After transfection with T7-RORγt and/or Flag-RSK2 expression vectors, cells were incubated for 1 day and lysed. Samples were then immunoprecipitated with anti-Flag M1 agarose affinity gel and examined by Western blotting with the antibodies indicated. (D) Luciferase reporter assay of the mIl17a promoter in 293T cells. After transfection with a β-gal expression vector, a mIl17a promoter luciferase vector, and/or expression vectors for RORγt, Rps6ka3, or Baz1b, 293T cells were cultured for 36 h and collected and luciferase assays were performed. As an internal control, ONPG-dependent β-gal activities were measured with a microplate reader. Each experiment was performed at least three times, and results are presented as means ± SD. *, P < 0.05. (E) Immunoprecipitation assays of DGCR14, RORγt, and RIP140 in 293T cells. After transfection with expression vectors for RORγt and/or Dgcr14, cells were incubated for 1 day and lysed. Samples were then immunoprecipitated with anti-ROR common and examined by Western blotting with the antibodies indicated. The asterisk indicates a nonspecific band. (F) Immunoprecipitation assays of DGCR14 and RORγt in 293T cells with or without T0901317. After transfection with expression vectors for RORγt and/or Dgcr14, cells were incubated for 1 day with or without 10 μM T0901317 and lysed. Samples were then immunoprecipitated with anti-DGCR14 and examined by Western blotting with the antibodies indicated.