1. A Constitutive NADPH Oxidase-Like System Containing gp91phox Homologs in Human Keratinocytes 
Walee. Chamulitrat, Wolfgang Stremmel, Tsukasa Kawahara, Kazuhito Rokutan, Hirotada Fujii, Kirstin Wingler, Harald H.H.W. Schmidt, Rainer Schmidt 
Journal of Investigative Dermatology 
Volume 122, Issue 4, Pages (April 2004)
DOI: /j X x
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Western blot analysis of the expression of NADPH oxidase cytosolic proteins in HaCaTs and GM16 cells. Western blotting was performed as described in Materials and Methods. (A) Rac1 protein was expressed in HaCaT, GM16, and positive controls brain and MHH cells. (B) p67phox protein was expressed in HaCaT, GM16, positive controls HL60, and MHH cells. (C) p47phox protein was expressed only in HL60 and MHH cells, but not in HaCaT and GM16 cells.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Western blot analysis of p40phox in cell lysates of HaCaTs and GM16 cells, and of p22phox in membranes of HaCaT cells. For experimental details see Materials and Methods. (A) p40phox protein was expressed in HaCaT, GM16, and positive control MHH cells. An additional positive control was recombinant histidine-tagged p40phox protein, which showed a heavier protein at 45 kDa indicated with an arrow. (B) p22phox protein was expressed in membranes of HaCaT and positive control undifferentiated HL60 cells.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
mRNA of Nox1, Nox2 and Nox4 in HaCaT and GM16 cells. Primers are shown in Table I with PCR methods described in Materials and Methods. (A) The expected 171 bp product of Nox1 from nested PCR was detected at the annealing temperature of 61°C in GM16 cells, and in positive control Caco-2 cells. Very faint almost undetectable 171 bp band was detected in HaCaT cells. Water control did not produce any PCR products. (B) The expected 550 bp product of Nox2 mRNA was detected at the annealing temperature of 62°C in HaCaT, GM16, and positive control HT29 cells. (C) The expected product of Nox4 mRNA was detected in GM16cells at the annealing temperature of 58°C.
Journal of Investigative Dermatology  , DOI: ( /j X x)
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5. Figure 4
Western blot analysis of Nox1 protein (63 kDa) in membranes of HaCaT, GM16, and positive control HT29 cells. (A) Nox1 protein (indicated at 63 kDa) was detected in membranes from HaCaT and GM16 cells (30 μg protein loaded). There were some weak protein bands due to unspecific reactivity of Nox1 antiserum. (B) The 63 kDa Nox1 protein was expressed in membranes of proliferating cells (HaCaT, sub, at ∼50% confluence) in higher amounts than those membranes of non-proliferating cells (HaCaT, con, at ∼85% confluence). The amount of protein loaded was 30 and 20 μg in HaCaT membranes in the left and right pair (HaCaT, con, and HaCaT, sub), respectively. HT29 membranes were loaded at 50 μg protein.
Journal of Investigative Dermatology  , DOI: ( /j X x)
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6. Figure 5
Specificity of anti-Nox1 antiserum demonstrated by GST-Nox1 fusion protein and the use of antigen peptide. (A) A recombinant GST protein of the cytosolic domain of human Nox1 (290–569 amino acids) was prepared. Nox1 antiserum recognized this recombinant protein, but not GST, as shown by western analysis (upper panel) and the protein staining (lower panel). (B) On the left-hand panel, the anti-Nox1 antiserum recognized the GST-Nox1 (loaded at 0.5 μg protein, left lane) as well as the 63 kDa Nox1 protein in T84 membranes (loaded at 20 μg protein, right lane). Fifty molar excess amounts of the antigen peptide completely absorbed the immunoreactivity of the Nox1-antiserum to the GST-Nox1 (left lane), and significantly decreased the expression of 63 kDa Nox1 protein in T84 membranes (right lane). On the right-hand panel, similar experiments were carried out using HaCaT membranes (loaded at 50 μg protein). The antigen peptide completely abolished the 63 kDa Nox1 protein signal detected in HaCaT membranes.
Journal of Investigative Dermatology  , DOI: ( /j X x)
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7. Figure 6
The reduced-minus-oxidized absorption difference spectra of solubilized neutrophil and HaCaT membranes. Solid dithionite was added prior to measuring difference spectra. Similar to neutrophil membranes, HaCaT membranes showed the Soret γ-band (at 426 nm) and α-band (at 558 nm).
Journal of Investigative Dermatology  , DOI: ( /j X x)
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8. Figure 7
Production of superoxide radicals detected by spin-trapping technique in a HaCaT cell-free system. The buffer used was 65 mM sodium phosphate, pH 7.0, containing 2 mM azide, 1 mM EGTA, and 65 μM DTPA. HaCaT membranes (90 μg per mL) and/or cytosol (98 μg per mL) were mixed in the presence of 65 mM DEPMPO. After addition of NADPH (500 μM), the incubation mixture was measured immediately. (A) HaCaT membranes were mixed with HaCaT cytosol in buffer containing DEPMPO and NADPH. (B) The same incubation as in (A) except that the cytosol was omitted. (C) The same incubation as in (A) but with addition of SOD (100 μg per mL). (D) The same incubation as in (A) except that membranes were omitted. (E) The same incubation as in (A) except that DPI (15 μM) was pre-incubated with membranes and cytosol 30 min prior to addition of NADPH. Spectrometer conditions were modulation amplitude, 1.2 G; microwave power, 20 mW; time constant, 20ms; scan rate, 107 G per min; 5 spectra with four-accumulated scans in each spectrum were subsequently added.
Journal of Investigative Dermatology  , DOI: ( /j X x)
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9. Figure 8
NADPH oxidase activity determined as rates of SOD-inhibitable cytochrome c reduction in a HaCaT cell-free system. HaCaT membranes (60 μg per mL), cytosol (80 μg per mL) were mixed with 40 μM AA in 65 mM phosphate buffer, pH 7.0, containing 170 mM sucrose, 2 mM azide, 1 mM EGTA, 20 μM GTP-γS and 80 μM cytochrome c. NADPH (200 μM) was added following a three-minute pre-incubation. Reference incubation was prepared in the presence of SOD (30 μg per mL). The absorbance at 550 nm of the sample and reference was measured in the first 5 min for initial rates. Membranes+cytosol showed increased activities compared with cytosol or membranes alone (*p<0.05, memb+cytosol vs cytosol or membranes alone). DPI inhibited oxidase activity in the memb+cytosol mixtures (§, p<0.05, memb+cytosol+DPI vs memb+cytosol).
Journal of Investigative Dermatology  , DOI: ( /j X x)
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