Flow cytometric determination of intracytoplasmic Wiskott–Aldrich syndrome protein in peripheral blood lymphocyte subpopulations

Kawai, S.; Minegishi, Masayoshi; Ohashi, Y; Sasahara, Yoji; Kumaki, Satoru; Konno, Tasuke; Miki, Hiroaki; Derry, Jonathan M.J.; Nonoyama, Shigeaki; Miyawaki, Toshio; Horibe, Keizo; Tachibana, Naoki; Kudoh, Emiko; Yoshimura, Yozo; Izumikawa, Yoshinori; Shinoda, Masahiro; Tsuchiya, Shigeru

doi:10.1016/s0022-1759(01)00549-x

Cited by 46 publications

(35 citation statements)

References 32 publications

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“…33 The mAb 5A5 stained WASP but not N-WASP on Western blots and did not cross-react with N-WASP. 33 The epitope recognized by 5A5 was localized to amino acids 146-265. WASP antibody W-485 was raised in rabbits immunized with amino acids 485-502 (C-terminus).…”

Section: Antibodiesmentioning

confidence: 88%

“…33,34 Cells were blocked with 5% normal goat serum (Vector Laboratories, Burlingame, CA) and reacted with 10 g/mL WASP 5A5 mAb or mouse IgG2a for 20 minutes on ice and washed twice with PBS, 1% fetal calf serum, 0.1% sodium azide, followed by FITC-labeled goat anti-mouse IgG2a antibodies (10 g/mL) for 20 minutes on ice. Cells were washed and analyzed on FACSCalibur (Becton Dickinson) immediately.…”

Section: Flow Cytometrymentioning

confidence: 99%

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Wiskott-Aldrich syndrome in a female

Lutskiy

Sasahara

Kenney

et al. 2002

Blood

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Section: Antibodiesmentioning

confidence: 88%

Section: Flow Cytometrymentioning

confidence: 99%

Wiskott-Aldrich syndrome in a female

Lutskiy

Sasahara

Kenney

et al. 2002

Blood

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“…FuGene transfection agent was from Roche (Indianapolis, IN). Generation of rabbit anti-WIP antiserum C14, anti-WASP antiserum K-374, and anti-WASP mAb 5A5 was described previously (5,6,37). Anti-WASP mAb B-9, anti-Vav, anti-CrkL mAb, and rabbit anti-NWASP antibody (H-100) were from Santa Cruz Biotechnology (Santa Cruz, CA).…”

Section: Methodsmentioning

confidence: 99%

WIP is a chaperone for Wiskott–Aldrich syndrome protein (WASP)

Fuente

Sasahara

Calamito

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

149

172

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Wiskott–Aldrich syndrome protein (WASP) is in a complex with WASP-interacting protein (WIP). WASP levels, but not mRNA levels, were severely diminished in T cells from WIP −/− mice and were increased by introduction of WIP in these cells. The WASP binding domain of WIP was shown to protect WASP from degradation by calpain in vitro . Treatment with the proteasome inhibitors MG132 and bortezomib increased WASP levels in T cells from WIP −/− mice and in T and B lymphocytes from two WAS patients with missense mutations (R86H and T45M) that disrupt WIP binding. The calpain inhibitor calpeptin increased WASP levels in activated T and B cells from the WASP patients, but not in primary T cells from the patients or from WIP −/− mice. Despite its ability to increase WASP levels proteasome inhibition did not correct the impaired IL-2 gene expression and low F-actin content in T cells from the R86H WAS patient. These results demonstrate that WIP stabilizes WASP and suggest that it may also be important for its function.

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“…For Western blotting, PBMC or NK cell preparations were lysed in 1% Nonidet P-40 lysis buffer and 20 g protein separated on a 10% polyacrylamide gel. Proteins were transferred to nitrocellulose membranes, which were incubated with murine anti-WASp mAb 5A5 (23). Bound antibody was detected with peroxidase-conjugated goat-anti-mouse (Jackson ImmunoResearch) and enhanced chemiluminescence detection system (Amersham Pharmacia).…”

Section: Methodsmentioning

confidence: 99%