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. Author manuscript; available in PMC: 2010 Feb 1.

Published in final edited form as: Arch Biochem Biophys. 2008 Dec 10;482(1-2):58–65. doi: 10.1016/j.abb.2008.11.028

Cells were grown to confluence under standard conditions either in 6 well plates or two-chamber culture slides. (A), Cells grown in 6 well plates were treated with 1 μM TFPI-2 and incubated at 37°C for the indicated times and harvested for fractionation. To detect the presence of offered proteins, cytosolic and nuclear fractions were probed with anti-TFPI-2 antibody as described in Methods. The purity of cytosolic and nuclear fractions was verified using anti-alpha-tubulin and anti-histone-H1 antibodies, respectively. For immunoblot analysis, the intensity of blot bands were assessed by densitometric semi-quantitation and depicted by a bar diagram (lower panel). (B), Cells grown in two-chamber culture slides were incubated with either vehicle control or 50 μg/ml of Alexa Fluor-conjugated TFPI-2 at 37°C for the indicated times and processed for confocal microscopy. (a), vehicle treated cells; (b), cells incubated with Alexa Fluor 488-conjugated TFPI-2 for 5 min; (c), cells incubated with Alexa Fluor 488-conjugated TFPI-2 for 10 min and (d), cells incubated with Alexa Fluor 488-conjugated TFPI-2 for 2 h. The nucleus is counterstained using DAPI in mounting media. (C), Another set of cells treated with 1 μM TFPI-2 were incubated at 4°C for the indicated times and processed for immunoblotting. For immunoblot analysis, the intensity of blot bands were assessed by densitometric semi-quantitation and depicted by a bar diagram (lower panel). (D), Cells were treated with 50 μg/ml of Alexa Fluor 488-conjugated TFPI-2, incubated at 4°C for the indicated times, and processed for confocal microscopy. (a), vehicle treated cells; (b), cells incubated with Alexa Fluor 488-conjugated TFPI-2 for 5 min; (c), cells incubated with Alexa Fluor 488-conjugated TFPI-2 for 10 min and (d), cells incubated with Alexa Fluor 488-conjugated TFPI-2 for 2 h. At 4°C, Alexa Fluor 488-conjugated TFPI-2 treated cells exhibited a punctate pattern of intracellular TFPI-2 distribution. [Arrows in panel (d) indicates the presence of the protein in the nucleus].

Cells were grown to confluence under standard conditions either in 6 well plates or two-chamber culture slides. (A), Cells grown in 6 well plates were treated with 1 μM TFPI-2 and incubated at 37°C for the indicated times and harvested for fractionation. To detect the presence of offered proteins, cytosolic and nuclear fractions were probed with anti-TFPI-2 antibody as described in Methods. The purity of cytosolic and nuclear fractions was verified using anti-alpha-tubulin and anti-histone-H1 antibodies, respectively. For immunoblot analysis, the intensity of blot bands were assessed by densitometric semi-quantitation and depicted by a bar diagram (lower panel). (B), Cells grown in two-chamber culture slides were incubated with either vehicle control or 50 μg/ml of Alexa Fluor-conjugated TFPI-2 at 37°C for the indicated times and processed for confocal microscopy. (a), vehicle treated cells; (b), cells incubated with Alexa Fluor 488-conjugated TFPI-2 for 5 min; (c), cells incubated with Alexa Fluor 488-conjugated TFPI-2 for 10 min and (d), cells incubated with Alexa Fluor 488-conjugated TFPI-2 for 2 h. The nucleus is counterstained using DAPI in mounting media. (C), Another set of cells treated with 1 μM TFPI-2 were incubated at 4°C for the indicated times and processed for immunoblotting. For immunoblot analysis, the intensity of blot bands were assessed by densitometric semi-quantitation and depicted by a bar diagram (lower panel). (D), Cells were treated with 50 μg/ml of Alexa Fluor 488-conjugated TFPI-2, incubated at 4°C for the indicated times, and processed for confocal microscopy. (a), vehicle treated cells; (b), cells incubated with Alexa Fluor 488-conjugated TFPI-2 for 5 min; (c), cells incubated with Alexa Fluor 488-conjugated TFPI-2 for 10 min and (d), cells incubated with Alexa Fluor 488-conjugated TFPI-2 for 2 h. At 4°C, Alexa Fluor 488-conjugated TFPI-2 treated cells exhibited a punctate pattern of intracellular TFPI-2 distribution. [Arrows in panel (d) indicates the presence of the protein in the nucleus].