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Fig. 1. Expression of WAVE family members in macrophages. (A) Total RNA was isolated from equivalent numbers of cells of the murine monocyte/macrophage RAW/LR5 cell line and primary murine thioglycollate-elicited peritoneal macrophages (M
) and RT-PCR was performed using sets of primers specific for the indicated transcripts. As a negative control the reverse transcription step was omitted (–RT). RNA from brain (Br) was used as a positive control for WAVE3 expression. (B) Western blot analysis of WASP, WAVE1 and WAVE2 protein expression in RAW/LR5 cells and bone marrow-derived macrophages (BMM) using isoform-specific rabbit polyclonal antibodies. Lysates from COS-7 cells transfected with either FLAG-tagged WASP (COS/WASP), FLAG-tagged WAVE1 (COS/W) or FLAG-tagged WAVE2 (COS/W2) were used as standards and probed with the indicated antibodies. (C) Corresponding quantitative analysis: WASP/WAVE over ß-actin signals in macrophage samples were quantified and expressed as a percentage of their corresponding standard (COS/W with W standing for WASP or WAVE1 or WAVE2). WASP/WAVE normalized intensities were subsequently compared between each other using the relative expression of standards determined by means of FLAG detection. Numbers were finally expressed as percentages of WASP expression in RAW/LR5 cells. n=3 different determinations on different lysates.
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