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. 1998 Feb 3;95(3):1289–1294. doi: 10.1073/pnas.95.3.1289

Figure 2.

Figure 2

Identification of a common 15-bp sequence crucial for PTPase, neuregulin, and Ras-dependent regulation of ɛ subunit promoter activity. (Left) Primary myotube cultures were cotransfected with a particular ɛ promoter/luciferase expression vector described in Fig. 1, CMV CAT (for normalization), with or without PTP CL100 expression vector. Cells were harvested 24 h after transfection for luciferase and CAT assays. (Middle) L6 cells were cotransfected with a particular ɛ promoter/luciferase expression vector and γ-722 CAT (for normalization). Twenty-four hours after the induction of differentiation, cells were treated with buffer or recombinant neuregulin (5 nM) for 60 h before harvesting and assaying for luciferase and CAT activities. (Right) Primary myotube cultures were cotransfected with a particular ɛ promoter/luciferase expression vector, γ-722 CAT (for normalization), with or without activated-Ras expression vector. Cells were maintained in low serum medium (DMEM/0.5% fetal calf serum) for 48 h before harvesting for luciferase and CAT assays. Experiments were repeated a minimum of three times. Bar graphs represent the average of triplicate transfections normalized to CAT activity; error bars represent ±SD. Dotted line indicates the control expression levels. Data are presented as a percent of control. Normalized luciferase activity for various constructs in the absence of cotransfection with a PTPase expression vector (control) was as follows: ɛ-5000, 207 (±14.6); ɛ-108, 22 (±3); ɛ-69, 477 (±87); ɛ3′Δ54, 949 (±187); ɛ3′Δ65, 1,165 (±82); ɛΔ(−56/−67), 1,090 (±144); ɛMUT1, 639 (±68); ɛMUT2, 1,042 (±63). Normalized luciferase activity for various constructs in the absence of treatment with neuregulin (control) was as follows: ɛ-5000, 7,778 (±1,070); ɛ-108, 481 (±29); ɛ-69, 2,517 (±162); ɛ3′Δ54, 11,057 (±961); ɛ3′Δ65, 36,451 (±6,110); ɛΔ(−56/−67), 22,076 (±1,557); ɛMUT1, 16,472 (±2,499); ɛMUT2, 18,273 (±3,677). Normalized luciferase activity for various promoter constructs in the absence of cotransfection with Ras expression vector (control) was as follows: ɛ-5000, 835 (±76); ɛΔ(−56/−67), 1,370 (±205); ɛ3′Δ65, 1,931 (±314); ɛMUT1, 1,368 (±31); ɛMUT2, 2,044 (±115).