Fibroblasts (1.5 × 105/mL) were pretreated with or without FP for 1 h prior to incubation in the presence or absence of nicotine for 48 h, washed with ice-cold PBS, and lysed in 1 mL of homogenization buffer (50 mmol/L NaCl, 50 mmol/L NaF, 50 mmol/L NaP2O7-10 H2O, 5 mmol/L ethylenediamine tetra-acetic acid [EDTA], 5 mM ethyleneglycol tetra-acetic acid, 2 mmol/L Na3 sodium [ortho] vanadate, 0.5 mmol/L phenylmethylsulfonyl fluoride, 0.01% Triton X-100, 10 μg/mL leupeptin, 10 mmol/L hydroxylethyl piperazine-ethanesulfonic acid, pH 7.4). Pretreatment with FP for 1 h prior to nicotine exposure was chosen because preliminary experiments revealed that concurrent treatment with FP showed inefficient inhibition. The resulting homogenate was centrifuged at 14,000 revolutions per minute for 5 min at 4°C. Protein concentration was determined by the Bradford method.13 The protein (100 μg) was mixed with an equal volume of 2 × sample buffer (125 mmol/L Tris HCl, pH 6.8, 4% sodium dodecysulfate [SDS], 20% glycerol, 5 to 10% β-mercaptoethanol, 0.004% bromophenol blue), boiled for 5 min, loaded onto a 5% SDS-polyacrylamide gel (10% SDS-polyacrylamide gel for CREB and phosphorylated CREB) with a 3.5% stacking gel, and electrophoresed for 2 h at 60 mA. The separated proteins were transferred onto nitrocellulose using a BioRad Trans Blot semidry transfer apparatus for 2 h at 1.0 mA/cm2, blocked with blotto (1 × Tris-buffered saline solution [10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl], 5% nonfat dry milk, 0.05% Tween-20) for 1 h at room temperature, and washed twice for 5 min with wash buffer (1 × Tris-buffered saline solution, 0.05% Tween-20). Blots were incubated with a polyclonal antibody raised against human fibronectin (1:1000 dilution), human CREB (1:1,000 dilution), or human phosphorylated CREB (Ser133) [1:1,000 dilution] for 24 h at 4°C, washed three times for 5 min with wash buffer, and incubated with a secondary rabbit antibody raised against goat IgG conjugated to horseradish peroxidase (1:15,000 dilution) for 1 h at room temperature. Identically loaded blots used for loading controls were incubated with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [1:2000 dilution] primary antibody. The blots were washed four times in wash buffer, transferred to freshly made ECL solution (Amersham; Arlington, IL) for 1 min, and exposed to radiograph film. Protein bands were quantified by densitometric scanning using a GS-800 calibrated laser densitometer (Bio-Rad; Hercules, CA). Western blots were repeated at least four times using samples from four separate experiments.