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. 2003 Dec 1;198(11):1689–1698. doi: 10.1084/jem.20031162

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Copyright © 2003, The Rockefeller University Press

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Figure 2. — LACK protein determinations and subcellular localization. (A) LACK protein expression in lack-targeted L. major lines. Lysates from 2 × 10⁷ lack-targeted L. major promastigotes were loaded, blotted, and probed with rabbit polyclonal antiserum raised against recombinant LACK protein (top). The blot was stripped and reprobed with goat polyclonal antiserum against eukaryotic 40S ribosomal protein S6 (bottom). Size markers (kD) are indicated. (B) Localization of GFP-LACK in L. major promastigotes. Stationary phase pL1SDGLKL1 transfectants were diluted 1 in 10 in PBS/0.02% formaldehyde. The parasites were viewed at ×1,000 with fluorescent excitation at 460–500 nm. Promastigote nucleus (N) and flagellum (Fl) are marked. (C) Localization of GFP in L. major promastigotes. Stationary phase pL1SDGL1 transfectants were treated and analyzed as described in B. (D) GFP-LACK protein expression in L. major lack++/−− transfectants. Parasites expressing GFP-LACK or GFP are indicated. Lanes 1 and 2, 2 × 10⁷ organisms; lanes 3 and 4, 2 × 10⁶ organisms. Location of endogenous LACK and GFP-LACK are indicated by large and small arrows, respectively.