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. 2010 Mar 11;285(19):14195–14209. doi: 10.1074/jbc.M109.052845

FIGURE 6.

FIGURE 6.

PP2A regulates FoxO3a on collagen matrix. A, serum-starved human lung fibroblasts were attached to collagen-coated plates (100 μg/ml) as a function of time and PP2A catalytic subunit expression levels were measured. Actin was used as a loading control. B, serum-starved lung fibroblasts were preincubated in the presence or absence of β1-integrin blocking antibody (1 μg/ml, BA) for 45 min. Cells were then attached to collagen-coated plates for 30 min. Western analysis was carried out to measure PP2A catalytic subunit expression (upper panel). PP2A activity was measured as described under “Materials and Methods” (lower panel). s/s, serum-starved cells; Un, untreated cells; BA, cells preincubated with β1-integrin blocking antibody; IgG, isotype control. C, lung fibroblasts infected with adenovirus expressing PP2A catalytic subunit or empty vector were serum-starved for 1 day. Cells were then attached to collagen-coated plates for 60 min. FoxO3a and HA-tagged proteins were then measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. PP2A, adenovirus expressing HA-tagged PP2A catalytic subunit; Vec, adenovirus expressing empty vector; Un, untreated cells; HA, HA-tagged antibody. D, serum-starved lung fibroblasts were preincubated with β1-integrin blocking antibody (1 μg/ml) in the presence or absence of 100 nm okadaic acid for 1 h. Cells were then attached to type I collagen for 30 min. FoxO3a expression levels were measured. GAPDH was used as a loading control. Un, untreated cells; P5D2, β1-integrin blocking antibody; DMSO, dimethyl sulfoxide control; OA, okadaic acid; IgG, isotype control. E, human lung fibroblasts were transfected with 10 nm PP2A siRNA (PP2A) or control siRNA (Control) along with untransfected (Un) cells followed by serum starvation for 1 day. Cells were then preincubated with β1-integrin blocking antibody (P5D2, 1 μg/ml) for 45 min and attached to collagen-coated plates for 30 min. FoxO3a and PP2A expression were then measured. Actin was used as a loading control.