Fig. 3.
Fig. 3. NF-Y participates in induction of JunB following IL-6 stimulation. / (A) Protein expression of NF-Y subunits. Extracts were derived from IL-6–treated M1 cells for the indicated times. Protein extracts (50 μg) were resolved on SDS-PAGE, using 10% acrylamide for NF-YA and 12.5% acrylamide for NF-YB and NF-YC expression. Western blots were prepared as described in “Materials and methods” and probed with antibodies. Antibodies against both NF-YA (1:1000) and NF-YB (1:500) were rabbit polyclonal and obtained from Rockland. NF-YC antibodies (1:1000) were goat polyclonal and obtained from Santa Cruz. (B,C) Establishment of M1 NF-YA DN cell lines. (B) RNA was extracted from several neo-resistance clones and analyzed by Northern blots, probing with NF-YA DN cDNA fragment. (C) Expression of NF-YA DN protein. TheNF-YA DN transgene encodes for the 43-kDa isoform. Westerns were performed as described earlier, probing with NF-YA antibodies (1:1000). (D) Expression of NF-YA DN inhibits NF-Y binding to −65/−52 IL-6RE DNA. EMSA experiments were performed with labeled JunBWT probe (10 000 cpm) and nuclear extracts (5 μg) from untreated cells. The cells used for the extracts are indicated above each lane. NF-Y binding was quantitated by using a Fuji BAS 2000 phosphoimage analyzer and software and expressed as a ratio in DN versus M1Neo9 cells (DN/M1Neo9). (E,F) NF-YA DN inhibits IL-6 induction of JunB. (E) XhoP2GD was transiently transfected into M1, M1Neo, and M1NF-YA DN cell lines, treated with or without IL-6 (100 ng/mL), CAT activity measured, and IL-6 induction determined. The SD of DN12 is too small to be seen. (F) JunB expression in M1Neo control and M1NF-YA DN IL-6–treated cells. RNA was extracted from the cell lines, untreated or treated with IL-6 (100 ng/mL) for the indicated time points, and Northern blots were probed with the appropriate labeled complementary DNA (cDNA) fragments. As in D, above, the level of expression was quantitated and expressed as a ratio to assess if NF-YA-DN suppresses JunB similarly to DNA binding. The suppression ratios of each were compared (JunB sup/NF-Y binding sup).

NF-Y participates in induction of JunB following IL-6 stimulation.

(A) Protein expression of NF-Y subunits. Extracts were derived from IL-6–treated M1 cells for the indicated times. Protein extracts (50 μg) were resolved on SDS-PAGE, using 10% acrylamide for NF-YA and 12.5% acrylamide for NF-YB and NF-YC expression. Western blots were prepared as described in “Materials and methods” and probed with antibodies. Antibodies against both NF-YA (1:1000) and NF-YB (1:500) were rabbit polyclonal and obtained from Rockland. NF-YC antibodies (1:1000) were goat polyclonal and obtained from Santa Cruz. (B,C) Establishment of M1 NF-YA DN cell lines. (B) RNA was extracted from several neo-resistance clones and analyzed by Northern blots, probing with NF-YA DN cDNA fragment. (C) Expression of NF-YA DN protein. TheNF-YA DN transgene encodes for the 43-kDa isoform. Westerns were performed as described earlier, probing with NF-YA antibodies (1:1000). (D) Expression of NF-YA DN inhibits NF-Y binding to −65/−52 IL-6RE DNA. EMSA experiments were performed with labeled JunBWT probe (10 000 cpm) and nuclear extracts (5 μg) from untreated cells. The cells used for the extracts are indicated above each lane. NF-Y binding was quantitated by using a Fuji BAS 2000 phosphoimage analyzer and software and expressed as a ratio in DN versus M1Neo9 cells (DN/M1Neo9). (E,F) NF-YA DN inhibits IL-6 induction of JunB. (E) XhoP2GD was transiently transfected into M1, M1Neo, and M1NF-YA DN cell lines, treated with or without IL-6 (100 ng/mL), CAT activity measured, and IL-6 induction determined. The SD of DN12 is too small to be seen. (F) JunB expression in M1Neo control and M1NF-YA DN IL-6–treated cells. RNA was extracted from the cell lines, untreated or treated with IL-6 (100 ng/mL) for the indicated time points, and Northern blots were probed with the appropriate labeled complementary DNA (cDNA) fragments. As in D, above, the level of expression was quantitated and expressed as a ratio to assess if NF-YA-DN suppresses JunB similarly to DNA binding. The suppression ratios of each were compared (JunB sup/NF-Y binding sup).

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