CD4 Cross-Linking (CD4XL) Induces RAS Activation and Tumor Necrosis Factor-α Secretion in CD4+ T Cells by Seetha M. Lakshmi Tamma, Narendra Chirmule, Hirosuka Yagura, Naoki Oyaizu, Vaniambadi Kalyanaraman, and Savita Pahwa Blood Volume 90(4):1588-1593 August 15, 1997 ©1997 by American Society of Hematology

CD4 ligation with gp160 or CD4XL with gp160 and anti-gp160 induces tyrosine phosphorylation of various cellular substrates: CD4+ T cells were treated with gp160 alone or with gp160 plus anti-gp160. CD4 ligation with gp160 or CD4XL with gp160 and anti-gp160 induces tyrosine phosphorylation of various cellular substrates: CD4+ T cells were treated with gp160 alone or with gp160 plus anti-gp160. Cell lysates were precleared and immunoprecipitated with antibodies to substrates p56lck, p59fyn, ZAP-70, PLC-γ1 (145 kD), and p95vav as indicated and immunoblotted with either the specific antibody or with antiphosphotyrosine antibody (4G10). Seetha M. Lakshmi Tamma et al. Blood 1997;90:1588-1593 ©1997 by American Society of Hematology

Activation of p21ras as measured by conversion of rasGDP to rasGTP following CD4XL. Activation of p21ras as measured by conversion of rasGDP to rasGTP following CD4XL. Labeling with 32 P-orthophosphate, cross-linking with gp160 and anti-gp160, cell lysing, and immunoprecipitation was performed as described in Materials and Methods. Ras bound GDP and GTP were eluted and analyzed by thin layer chromatography, visualized by autoradiography, and quantitated by densitometry. Conversion of rasGDP to rasGTP was calculated as a ratio of percent rasGTP/GDP:GTP. Data represents mean of two experiments. Seetha M. Lakshmi Tamma et al. Blood 1997;90:1588-1593 ©1997 by American Society of Hematology

CD4 cross-linking with gp160 and anti-gp160 in dominant negative ras mutant-transfected cells results in decreased TNF-α secretion. CD4 cross-linking with gp160 and anti-gp160 in dominant negative ras mutant-transfected cells results in decreased TNF-α secretion. CD4+ Jurkat T cells were transiently transfected with the dominant negative ras mutant (rasN17) or control vector by electroporation as described in Materials and Methods. Concentration of plasmid DNA used for transfection (μg/mL) is indicated. CD4XL was performed with gp160 and anti-gp160 antibody. TNF-α in the culture supernatants was measured by ELISA. Mock-medium (▪), Mock-CD4XL (♦), RasN17-medium (•), RasN17-CD4XL (▴). Seetha M. Lakshmi Tamma et al. Blood 1997;90:1588-1593 ©1997 by American Society of Hematology