(A) Rescue of INT6 siRNA effect by expression of a resistant form of INT6. The experiment was performed as described in the legend to Figure 4 with the PSL-H4 reporter construct and the control or I6.4 siRNA duplexes. The latter matches with a sequence of the 3′ UTR of the INT6 mRNA. Together with the luciferase constructs, cells were also cotransfected with a control or an INT6 expression vector lacking the 3′ UTR. Luciferase activity was measured and is represented as described in the legend to Figure 4. (B) Extracts of cells used for the experiment shown in panel A were analyzed by immunoblot using an antibody to INT6 (top panel) or to β-actin (bottom panel). The position of the signal corresponding to these proteins is indicated. (C) The experiment described in panel A was also performed with the PSL-H2B reporter construct. Results are represented as in A. (D) Inhibitory effect of overexpression of a C-terminally truncated INT6 mutant on PSL-H4 activity. 0.3 × 105 HeLa cells were transfected with the PSL-H4 reporter together with either an empty vector or 4 μg and 8 μg of a construct expressing an INT6 mutant corresponding to the N-terminal 276 amino acids. Luciferase activity was measured and is represented as described in the legend to Figure 4. (E) Extracts of cells used for the experiment shown in panel C were analyzed by immunoblot using an antibody directed against the N-terminal 19 amino acids of INT6 (top panel) or to β-actin (bottom panel). The position of the signal corresponding to INT6, INT6ΔC, and β-actin is indicated. The signals corresponding to INT6 and INT6ΔC were quantified, and numbers below the blot are the fractions of INT6ΔC with respect to INT6.
