Proteasome-dependent regulation of Syk tyrosine kinase levels in human basophils  Lama A. Youssef, B Pharm, Bridget S. Wilson, PhD, Janet M. Oliver, PhD  Journal of Allergy and Clinical Immunology  Volume 110, Issue 3, Pages 366-373 (September 2002) DOI: 10.1067/mai.2002.127562 Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 1 Proteasome and caspase inhibitors increase Syk protein levels in releaser and nonreleaser basophils. A, Top: Basophils (>99% pure) were incubated with DMSO or with PSI (60 μmol/L), calpain inhibitor V (50 μmol/L), or caspase inhibitor III (25 μmol/L) for 4 hours. Solubilized proteins (2 × 105 cell equivalents/lane) were resolved by SDS-PAGE and analyzed by Western blotting through use of anti-Syk and anti-Lyn antibodies. Bottom: Densitometric analysis of gels shown above. Values are presented as arbitrary (Arb) densitometric units. B, Basophils were incubated with DMSO or with PSI (60 μmol/L), Lactacystin (40 μmol/L), or ALLN (50 μmol/L) for 3 hours, then analyzed by Western blotting through use of anti-Syk, anti-Lyn, and anti-actin antibodies. C, Nonreleaser basophils were incubated with PSI (60 μmol/L) for 30 minutes and 1, 2, and 3 hours, then analyzed through use of anti-Syk antibodies. D, Longer exposure of the films in Fig 1, C . E, Western blot analysis was performed as above through use of anti-Cbl mAbs and then through use of anti-actin mAbs. Each experiment was performed at least 3 times. Journal of Allergy and Clinical Immunology 2002 110, 366-373DOI: (10.1067/mai.2002.127562) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 2 In vitro ubiquitination of Syk. A, Protein A-Sepharose-bound Syk protein was immunopurified from 900 μg aliquots of unstimulated basophil lysates. Immunoprecipitates were subjected to in vitro ubiquitination reactions for 2 hours through use of RRL with added ubiquitin (Ub) and ubiquitin aldehyde (Ub-Ald) . Syk immunoprecipitates were eluted with SDS-sample buffer, resolved by 8% SDS-PAGE, and immunoblotted with anti-Syk mAb (top panel) and then with anti-Ub (bottom panel) . B, Anti-Syk immunoprecipitates from 600-μg aliquots of basophil lysates were used in the assay. The reactions were carried out for 30 minutes to 2 hours in the presence or absence of PSI. Western blot analysis was performed as above. Molecular weight markers (in kilodaltons) are provided on the left. The same results were obtained in 2 separate experiments. Journal of Allergy and Clinical Immunology 2002 110, 366-373DOI: (10.1067/mai.2002.127562) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 3 Effects of protease inhibitors on Syk levels in lymphocytes and monocytes. A, Monocytes (98% pure) and total lymphocytes (>85% pure) were incubated with DMSO (lane 1) or PSI (60 μmol/L) for 1, 2, or 3 hours (lanes 2, 3, and 4, respectively) or with calpain inhibitor V (50 μmol/L; lane 6 ) or caspase inhibitor III (25 μmol/L; lane 7 ) for 3 hours. Proteins from 2 × 105 cells/lane were fractionated by SDS-PAGE, and Western blot analysis was performed as described in the legend to Fig 1. B, CD4+CD45RO− T cells were incubated for 3 hours in the absence or presence of PSI (60 μmol/L). Extracts from 3.5 × 105 cells were analyzed by Western blotting through use of anti-Syk and anti-actin antibodies. Results are typical of 3 replicate experiments. Journal of Allergy and Clinical Immunology 2002 110, 366-373DOI: (10.1067/mai.2002.127562) Copyright © 2002 Mosby, Inc. Terms and Conditions