Figure 6.
Figure 6. Binding of RARα to RAREs and RA-dependent gene transcription via RAREs is mTOR independent, but PI3′K sensitive. (A) MCF-7 cells were transfected with a RARE-luciferase construct. Forty-eight hours after transfection, cells were treated for 60 minutes in the presence or absence of rapamycin (20 nM). The cells were then incubated overnight with or without RA (1 μM), in the continuous presence or absence of rapamycin, and luciferase activity was measured. The data are expressed as fold increase in luciferase activity over RA-untreated control samples for each condition, normalized for β-galactosidase activity. Means plus or minus SE values of 3 independent experiments are shown. (B) MCF-7 cells were transfected with a RARE-luciferase construct. Forty-eight hours after transfection, cells were treated for 60 minutes in the presence or absence of LY294002 (50 μM). The cells were then incubated overnight with or without RA (1 μM), in the continuous presence or absence of LY294002, and luciferase activity was measured. The data are expressed as fold increase in luciferase activity over RA-untreated control samples for each condition, normalized for β-galactosidase activity. Means plus or minus SE values of 3 independent experiments are shown. (C) NB-4 cells were incubated with RA in the presence or absence of the PI3′K inhibitor LY294002. Equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of PKC-δ at Thr505. (D) The same blot shown in panel C was stripped and reprobed with an anti-PKC-δ antibody, to control for protein loading. (E) NB-4 cells were treated with RA in the presence or absence of the PI3′K inhibitor wortmannin (50 nM) or the PKC-δ inhibitor rottlerin (10 μM). Equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with antibody against the phosphorylated form of p70 S6K. (F) The blot shown in panel E was stripped and reprobed with an anti-p70 S6 kinase antibody. (G) Similar experiment to the one shown in panel E, except that immunoblotting was performed using an anti-phospho-235/236-ribosomal S6 protein (rpS6) antibody. (H) The blot shown in panel G was stripped and reprobed with anti-rpS6 antibody.

Binding of RARα to RAREs and RA-dependent gene transcription via RAREs is mTOR independent, but PI3′K sensitive. (A) MCF-7 cells were transfected with a RARE-luciferase construct. Forty-eight hours after transfection, cells were treated for 60 minutes in the presence or absence of rapamycin (20 nM). The cells were then incubated overnight with or without RA (1 μM), in the continuous presence or absence of rapamycin, and luciferase activity was measured. The data are expressed as fold increase in luciferase activity over RA-untreated control samples for each condition, normalized for β-galactosidase activity. Means plus or minus SE values of 3 independent experiments are shown. (B) MCF-7 cells were transfected with a RARE-luciferase construct. Forty-eight hours after transfection, cells were treated for 60 minutes in the presence or absence of LY294002 (50 μM). The cells were then incubated overnight with or without RA (1 μM), in the continuous presence or absence of LY294002, and luciferase activity was measured. The data are expressed as fold increase in luciferase activity over RA-untreated control samples for each condition, normalized for β-galactosidase activity. Means plus or minus SE values of 3 independent experiments are shown. (C) NB-4 cells were incubated with RA in the presence or absence of the PI3′K inhibitor LY294002. Equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of PKC-δ at Thr505. (D) The same blot shown in panel C was stripped and reprobed with an anti-PKC-δ antibody, to control for protein loading. (E) NB-4 cells were treated with RA in the presence or absence of the PI3′K inhibitor wortmannin (50 nM) or the PKC-δ inhibitor rottlerin (10 μM). Equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with antibody against the phosphorylated form of p70 S6K. (F) The blot shown in panel E was stripped and reprobed with an anti-p70 S6 kinase antibody. (G) Similar experiment to the one shown in panel E, except that immunoblotting was performed using an anti-phospho-235/236-ribosomal S6 protein (rpS6) antibody. (H) The blot shown in panel G was stripped and reprobed with anti-rpS6 antibody.

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