Journal: Infection and Immunity

Article Title: The Absence of Hck, Fgr, and Lyn Tyrosine Kinases Augments Lung Innate Immune Responses to Pneumocystis murina

doi: 10.1128/iai.01441-08

Figure Lengend Snippet: FIG. 1. P. murina activates SFKs in alveolar macrophages. Alveolar macrophages were isolated from 6- to 8-week-old, male C57BL/6 mice and stimulated for 10 to 90 min with P. murina (PC) at a ratio of macrophages to total P. murina cells of 1:100. The controls included alveolar macrophages cultured in medium alone (Unstim). After this, cell lysates were extracted, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PVDF membranes, and immunoblotted with anti-pHck 411 or rabbit anti-pLyn 507 IgG. Positive bands were identified using an ECL Western blot detection kit and were subsequently analyzed using Image J software (NIH). The blots are representative blots for (A) p-Lyn and (B) p-Hck levels in unstimulated and P. murina-stimulated alveolar macrophages. Cumulative data from three independent studies employing Image J software were used to determine the area under the curve values for (C) p-Lyn and (D) p-Hck and their beta-actin controls for unstimulated and P. murina-stimulated alveolar macrophages. The data are expressed both as the ratio of p-Lyn or p-Hck to actin and as the change after unstimulated samples were normalized to a value of 1. Data are the means standard errors of the means. ** and ***, P 0.01 and P 0.001, respectively (paired two-tailed Student’s t test).

Article Snippet: After this, the PVDF membrane was incubated with goat polyclonal anti-pHck 411 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-pLyn 507 IgG (Cell Signaling Technologies, Danvers, MA), or chicken anti-beta actin IgY (Novus Biologics) in 2% skim milk (all primary antibodies were used at a dilution of 1:1,000 for 2 h at room temperature), followed by horseradish peroxidase-conjugated donkey anti-goat IgG, horseradish peroxidase-conjugated goat anti-rabbit IgG, or horseradish peroxidase-conjugated rabbit anti-chicken IgY (all at a dilution of 1:2,000 for 2 h at room temperature).

Techniques: Isolation, Cell Culture, Polyacrylamide Gel Electrophoresis, Western Blot, Software, Two Tailed Test