1. Biochemical Characterization of S100A2 in Human Keratinocytes: Subcellular Localization, Dimerization, and Oxidative Cross-Linking1 
Rohini Deshpande, Timothy L. Woods, Jian Fu, Tong Zhang, Stefan W. Stoll 
Journal of Investigative Dermatology 
Volume 115, Issue 3, Pages (September 2000)
DOI: /j x
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Subcellular localization of S100A2 in NHK. (A) Lack of S100A2 membrane association in NHK. Hypotonic lysates were subjected to × g centrifugation followed by SDS–PAGE and western blotting. Note that the × g pellets lack S100A2 and that addition of nonionic detergent to the lysis buffer did not increase the amount of S100A2 recovered in the supernatant. (B) S100A2 is not associated with the cytoskeleton in NHK. Digitonin lysates were analyzed by SDS–PAGE followed by western blotting. Lane a, digitonin-soluble fraction; lane b, digitonin wash; lane c, digitonin-insoluble fraction. Equal aliquots of a plate were loaded on to each lane; amounts of protein loaded were different in each lane. (C) Digitonin-insoluble S100A2 is localized to nuclei. Immunofluorescence microscopy of S100A2 (upper panels) or S100A10 (lower panels) in NHK fixed with 4% paraformaldehyde after rocking for 5 min at 4°C in phosphate-buffered saline (left panels) or digitonin in import buffer (right panels). Note the presence of nuclear staining and the absence of digitonin-insoluble extranuclear staining for S100A2 (upper panels), as opposed to the lack of nuclear staining and presence of digitonin-insoluble extranuclear staining for S100A10 (lower panels).
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Homodimerization of S100A2. (A) Epitope-tagged S100A2 efficiently homodimerizes in an immunoprecipitation/western blot assay. Western blots of Tris–glycine SDS–PAGE gels are shown. Experimental conditions (described in Materials and Methods) are indicated above and to the left of the autoradiographs. Note that immunoprecipitation with anti-FLAG results in the pulldown of HA-tagged S100A2. (B) S100A2 forms a homodimer in living cells (yeast two-hybrid assay). Various constructs were grown in selective (left panel) or nonselective medium (right panel) as described in Materials and Methods. Note that only the yeast strain transfected with the S100A2 plasmid is capable of growing in selective media.
Journal of Investigative Dermatology  , DOI: ( /j x)
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4. Figure 3
Presence of disulfide-linked S100A2 dimers in nondenaturing extracts of NHK. Lanes a–d contain digitonin lysates of NHK prepared in the absence of reducing agents, as described in Materials and Methods. All procedures were carried out at 4°C. Tris–glycine PAGE gels were run within 1 h of cell lysis. The gel producing lanes a and b was run without SDS in the loading buffer. Samples were loaded using Laemmli loading buffer without denaturant (SDS) or reducing agent (DTT) (lane a); without SDS, but with DTT (lane b); with SDS, but without DTT (lane c); or with both SDS and DTT (lane d). The molecular weight markers shown to the right of the autoradiograms apply only to lanes c and d.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Lack of disulfide-linked S100A2 dimers in ionic detergent extracts of NHK, and appearance of oxidized S100A2 dimers after H2O2 treatment of intact cells. (A) Comparison of various extraction buffers. Untreated control NHK cultures are compared with cultures treated with 1 m M H2O2 for 90 min prior to extraction. Western blots of Nu-PAGE gels decorated with S100A2 MoAb (Sigma) were prepared as described in Materials and Methods. Extraction conditions were as follows: lane 1, cells were lysed by rocking at 4°C in 1× LDS buffer. These lysates were loaded directly. Lane 2, cells were lysed by rocking at 4°C in hypotonic lysis buffer (RSB) containing 2% LDS. These lysates were adjusted to approximately the composition of 1× LDS buffer prior to loading. Lane 3, cells were allowed to swell for 5 min in hypotonic lysis buffer at 4°C followed by scraping and Dounce homogenization. Lane 4, cells were lysed by rocking at 4°C in import buffer plus 50 μg per ml digitonin. In lanes 3 and 4, one-fourth volume of 4× LDS buffer was added to the lysates prior to loading. Results are shown from two of five keratinocyte strains tested. All five strains were derived from different donors; all strains yielded essentially identical results. (B) Time course of H2O2-induced oxidation. Intact NHK were incubated with 1 m M H2O2 for the times shown at the top of the figure. The experiment shown is representative of three independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
Exposure of oxidized S100A2 dimers to high calcium concentrations causes formation of cross-linked aggregates of S100A2. (A) Calcium effects on pre-oxidized dimers. Calcium was added to a nondenaturing digitonin lysate of NHK at various concentrations as listed at the top of the figure. Note that the lysates consisted primarily of oxidized S100A2 dimers (open triangles) prior to calcium treatment. Solid triangles indicate mobility of monomeric S100A2. In the left panel, the loading buffer lacked DTT. In the right panel, the loading buffer contained 37.5 mM DTT. Hypotonic lysates prepared using RSB yielded very similar results (data not shown). Also, addition of 1 mM EGTA to nondenaturing extracts of NHK had no detectable effect on the S100A2 band pattern, suggesting that calcium is not required for maintenance of the S100A2 dimer (data not shown). (B) Lack of calcium effect in intact cells. Intracellular calcium levels were artificially elevated by addition of the concentrations of CaCl2 indicated at the top of the figure to NHK together with 3 μM of the calcium ionophore A Denaturing extracts were then prepared using 1× LDS buffer as described in Materials and Methods. The lane labeled ‘‘H2O2’' represents a positive control for dimerization. This lane contained a denaturing extract of intact NHK that had been treated with 1 mM H2O2 for 90 min prior to extraction.
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7. Figure 6
Five different anti-S100A2 antibodies detect similar S100A2 HMW band patterns. Western blots of replicate Nu-PAGE gels decorated with five different anti-S100A2 antibodies are shown. Lanes 1 and 4, digitonin lysates from untreated cells. Lanes 2 and 5, digitonin lysates from cells treated with 1 mM hydrogen peroxide for 1.5 h. Lanes 3 and 6, calcium chloride was added to digitonin lysates from untreated cells, to a final concentration of 1 mM. In lanes 1–3, DTT was omitted from the in loading buffer. In lanes 4–6, 37.5 mM DTT was present in the loading buffer. (A) MoAb from Transduction Labs; (B) MoAb from Sigma; (C) MoAb from DAKO; (D) rabbit polyclonal antibody from DAKO; (E) rabbit polyclonal antibody, gift from DR Claus Heizmann. Open triangles indicate mobility of S100A2 oxidized dimer; closed triangles indicate mobility of monomeric S100A2.
Journal of Investigative Dermatology  , DOI: ( /j x)
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