Desmosome Assembly and Keratin Network Formation After Ca2+/Serum Induction and UVB Radiation in Hailey–Hailey Keratinocytes  Markus Bernards, Bernhard P. Korge  Journal of Investigative Dermatology  Volume 114, Issue 5, Pages 1058-1061 (May 2000) DOI: 10.1046/j.1523-1747.2000.00960-2.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Lysis-buffer with NP-40 provides most suitable coimmunoprecipitation conditions. 35S-Met/Cys-labeled keratinocytes were lyzed over night at 4°C in buffers containing NP-40 (modified protocol fromOursler et al. 1992) or Triton X-100 (modified protocol fromKowalczyk et al. 1994), or in SDS-lysate-buffer for 10 min at 95°C. After immunoprecipitation with plakoglobin-antibodies PG5.1 (Progen Biotechnik, Heidelberg, Germany) (lane 1) and γC-15 (Transduction Laboratories, Lexinton, GB) (lanes 2–6) precipitates were washed with 0.3% NP-40 (lanes 1, 2), 0.1% SDS (lane 3), 0.5% SDS (lane 4), 0.3% Triton X-100 (lane 5), or high/low salt wash buffers (lane 6) and separated by SDS-PAGE. For specificity of coimmunoprecipitations compare preclearing without specific antibody (pre). Left side: molecular weight marker in kDa. Immunoprecipitation was performed as follows: Keratinocytes were scraped off in 600 μl NP-40 lysis buffer (0.5% Nonidet p 40 in TBS; 20 mM Tris-HCl, pH 7.6, 137 mM NaCl) with protease inhibitors (20 μg aprotinin per ml, 20 μg leupeptin per ml, 120 μg pefabloc per ml, 350 μg trypsin-inhibitor per ml, 2 μg pepstatin A ethanol per ml, 50 μg antipain per ml, final concentrations in 1 ml) or in 600 μl Triton lysis buffer (1% Triton X-100 in TBS with protease inhibitors). After lysis the lysate was diluted with 400 μl TBS and centrifuged at 100,000 × g for 30 min. For quantitative immunoprecipitations equal amounts of total radioactivity were used. The lysate was precleared with protein-G-sepharose (PGS) and then with an RαM-antibody coupled to PGS. Immunoprecipitation was performed with 3 μg antibody coupled to PGS. Alternatively, keratinocytes were lyzed in 100 μl SDS-lysate-buffer (10 min 95°C, SDS lysis buffer: 1% SDS, 5 mM EDTA, 2 mM EGTA, 0.1 mM DTT, 10 mM Tris pH 7.5, protease inhibitors) according to a protocol kindly provided by Kathleen Green (Chicago, IL) and diluted with 900 μl dilution buffer (5 mM EDTA, 2 mM EGTA, 1% Triton X-100, 1% Na-desoxychelate, 0.1% SDS, 120 mM NaCl, 25 mM KCl, 0.1 mM DTT, 15 mM Tris pH 7.5, protease inhibitors as above). Immunoprecipitation was performed as described for NP-40. After immunoprecipitation the PGS was washed one time each in dilution buffer, in high salt buffer (dilution buffer with 1 M NaCl) and low salt buffer (10 mM Tris pH 7.5, 2 mM EDTA, 0.1 mM DTT, protease inhibitors). Journal of Investigative Dermatology 2000 114, 1058-1061DOI: (10.1046/j.1523-1747.2000.00960-2.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Identification of coimmunoprecipitated proteins using γC-15 plakoglobin antibody. γC-15-immunoprecipitate was separated by SDS-PAGE, transferred to a nylon-membrane and subsequently incubated with antibodies against plakoglobin (Pg, antibody: γC-15), α-catenin (+α), β-catenin (+β), desmoglein (+Dsg), and E-cadherin (E-Cad) without removing the antibodies after the incubation steps. ECL detection of these primary antibodies was performed by using a peroxidase-coupled secondary antibody. Note the detection of heavy and light chain of the immunoprecipitation antibody by this secondary antibody (approximately 50 kDa and 25 kDa). Compare detected proteins in the immunoprecipitate with a total protein extract of primary keratinocytes (total prot.) incubated in the same way. Left side: molecular weight marker in kDa. Journal of Investigative Dermatology 2000 114, 1058-1061DOI: (10.1046/j.1523-1747.2000.00960-2.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Influence of UV-B radiation on desmosome formation and keratin network reorganization. After UVB radiation with 300 mJ per cm2 HH and control keratinocytes were subjected to a calcium/serum shift (Ca/FCS), fixed after 3 and 24 h and stained with antibodies against desmoplakin (Dp1 + 2) and pan-keratin (keratin). Keratin network reorganization showed whorl-like perturbation after 3 h (arrows), whereas desmosomal proteins appeared intracellularly after 24 h. Scale bar: 30 μm. Journal of Investigative Dermatology 2000 114, 1058-1061DOI: (10.1046/j.1523-1747.2000.00960-2.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions