Fig. 3.

Inhibition of NF-κB by CinF requires residues important for the putative fructose 1,6-bisphosphate phosphatase activity. (A) Tyr362 is important for the activity of CinF. HEK293T cells were transfected with combinations of plasmids, and the induction of NF-κB was determined after PMA treatment (Upper). The expression of Flag-CinF and its mutant was probed, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control (Middle and Lower). The relative intensity of the bands representing p-IκBα or IκBα was quantitated using the bands of GAPDH as reference. Data shown were from three independent experiments. (B and C) Inhibition of p65 nuclear translocation by CinF requires Tyr362. Samples were prepared and processed as described in Fig. 2B, and the rates of nuclear p65 were scored from samples transfected to express CinF or its mutants. At least 300 cells were counted for each sample. Results shown were from the average of three independent experiments (B). Representative images of samples from one experiment (C). (Scale bar: 5 μm.) Statistical analysis was performed by Student’s t test.