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. 2003 Jul;109(3):351–359. doi: 10.1046/j.1365-2567.2003.01671.x

Figure 1.

Figure 1

Tyrosine phosphorylation of a 38 000-molecular weight (MW) protein in cells of mouse progenitor B (pro-B) and precursor B (pre-B) cell lines. (a) (panel A) 1xN/2b (pre-B) and WEHI231 (immature B) cells were stimulated with 5 µg/ml of goat F(ab′)2 anti-mouse immunoglobulin M (IgM) antibody for 2 min at 37°. Whole-cell lysates were fractionated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (10% gel) and transferred to nitrocellulose membrane. Tyrosine-phosphorylated proteins were detected by blotting with the PY20 monoclonal antibody (mAb). (a) (panel B) 1xN/2b cells were stimulated with 5 µg/ml of goat F(ab′)2 anti-mouse IgM antibody for 2 min at 37°. Whole-cell lysates were immunoprecipitated with rabbit anti-mouse LAT antibodies. Immunoprecipitates were fractionated by SDS–PAGE (10% gel), transferred to nitrocellulose membrane and immunoblotted with the PY20 mAb. (b) Cells of 1xN/2b (pre-B), DIF9 (pro-B), 5.7.10 (pre-B) and DW34 (pro-B) were stimulated with 5 µg/ml goat F(ab′)2 anti-mouse IgM antibody for 5 min at 37°. Whole-cell lysates were fractionated by SDS–PAGE (10% gel) and transferred to nitrocellulose membranes. Tyrosine-phosphorylated proteins were detected by blotting with the PY20 mAb. (c) DIF9 cells were preincubated with biotin-conjugated anti-Igβ antibody for 5 min on ice, followed by the addition of avidin (0, 10 or 100 µg/ml) and further incubation at 37° for 2 min. Whole-cell lysates were immunoblotted with the PY20 mAb. (d) LAT protein was detected by Western blot analysis of whole-cell lysates from the indicated cell lines. Lane 1, 1xN/2b (pre-B); lane 2, Jurkat (human T cell); lane 3, DIF9 (pro-B); lane 4, 5.7.10 (pre-B); lane 5, 70z/3 (late pre-B); lane 6, WEHI231 (immature B). (e) Reverse transcription–polymerase chain reaction (RT–PCR) to detect LAT was performed with total RNA prepared from the indicated mouse cell lines. Lane 1, DIF9 (pro-B); lane 2, 1xN/2b (pre-B); lane 3, 70z/3 (late pre-B); lane 4, WEHI231 (immature B); lane 5, EL4 (T cell). The results were confirmed in a separate experiment. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.