Protein C Inhibitor is Expressed in Keratinocytes of Human Skin

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Protein C Inhibitor is Expressed in Keratinocytes of Human Skin Michael Krebs, Pavel Uhrin, Anja Vales, Maria J. Prendes-Garcia, Johann Wojta, Margarethe Geiger, Bernd R. Binder Journal of Investigative Dermatology Volume 113, Issue 1, Pages 32-37 (July 1999) DOI: 10.1046/j.1523-1747.1999.00644.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 PCI antigen is present in normal human skin. Frozen sections of normal human skin were stained with monoclonal anti-PCI IgG (4PCI) (A) or nonimmune mouse IgG (B) as described in Materials and Methods. Scale bar: 50 μm. Journal of Investigative Dermatology 1999 113, 32-37DOI: (10.1046/j.1523-1747.1999.00644.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 HaCaT, A431, and HepG2 cells produce PCI mRNA. Total RNA obtained from HaCaT (1 μg, lane B), A431 (1 μg, lane C), and HepG2 (0.2 μg, lane D and 0.1 μg, lane E) cells was subjected to reverse transcription–polymerase chain reaction using primers specific for a 442 bp fragment of PCI-cDNA as described in Materials and Methods. The fragments obtained were loaded on a 1% agarose gel and visualized after electrophoresis by ethidium bromide staining. The control in lane F is a template free reaction. Lane A contains molecular weight markers (λEcoRI–HindIII digest). Journal of Investigative Dermatology 1999 113, 32-37DOI: (10.1046/j.1523-1747.1999.00644.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 (A) PCI antigen is present in conditioned media from keratinocytes. Conditioned media from HaCaT (lanes A and B) and A431 (lanes C and D) cells were collected after incubation times of 24 h (lanes A and C) or 48 h (lanes B and D). Conditioned media (24 h) from HepG2 cells (lane E) were used as positive control. The samples were run on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred on to a PVDF membrane. Rabbit anti-PCI IgG and peroxidase linked anti-rabbit antibodies were used to identify PCI related protein bands as described in Materials and Methods. (B) PCI antigen accumulates in conditioned media of HaCaT, A431, and HepG2 cells. Conditioned media were collected from confluent monolayers after incubation times of 4, 8, and 24 h and PCI antigen was determined by a specific ELISA. Data presented are the mean values of two determinations. Journal of Investigative Dermatology 1999 113, 32-37DOI: (10.1046/j.1523-1747.1999.00644.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 HaCaT cells secrete uPA, tPA, and similar amounts of PCI and PAI-1. Conditioned media were collected from confluent monolayers after incubation times of 4, 8, and 24 h and the concentrations of the respective antigens were determined by specific ELISA. Journal of Investigative Dermatology 1999 113, 32-37DOI: (10.1046/j.1523-1747.1999.00644.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Proliferating and nonproliferating HaCaT cells express similar amounts of intracellular PCI. PCI antigen was detected in permeabilized disperse growing and confluent cells using rabbit anti-PCI IgG (thick line). The thin line represents the fluorescence of control cells treated only with the second, fluorescein-labeled antibodies. In panel A the pattern of fluorescence of confluent, growth arrested cells and in panel B the intensity of fluorescence of scattered, proliferating cells is shown. The FACS analyzes for permeabilized cells are shown on the left and the ones for nonpermeabilized cells are presented on the right. Journal of Investigative Dermatology 1999 113, 32-37DOI: (10.1046/j.1523-1747.1999.00644.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions