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. 2007 Jul 13;6(9):1646–1655. doi: 10.1128/EC.00177-07

FIG. 6.

FIG. 6.

(A) Schematic representation of constructs encoding full-length and truncated FLAG-tagged EhLimA. (a) N-terminus-tagged full-length EhLimA (FLAG-EhLimA); (b) N-terminus-tagged EhLimA lacking half of the N terminus LIM domain (N-TRUNC); (c) N-terminus-tagged EhLimA lacking the glutamic acid-rich C terminus (C-TRUNC); (d) C-terminus-tagged full-length EhLimA (C-FLAG). (B) Protein expression of FLAG-EhLimA is confirmed in a Western blot analysis using anti-EhLimA antibodies (lanes I) and anti-FLAG monoclonal antibodies (lanes II). Lane 1 and 2 contain total cell lysates of wild-type cells and FLAG-EhLimA-overexpressing cells, respectively. (C) Different amounts of total cell lysates of FLAG-EhLimA-overexpressing cells (lanes 1 to 3) and wild-type cells (lane 5) were incubated with anti-FLAG monoclonal antibodies covalently attached to agarose. Immunoprecipitated FLAG-EhLimA was analyzed by gel electrophoresis followed by total protein staining of the gel (top gel). Coimmunoprecipitation of actin was analyzed in a Western blot of immunoprecipitated protein products using antiactin antibodies (bottom gel). Lane 1, 250 μg total lysate; lane 2, 100 μg total lysate; lane 3, 50 μg total lysate; lane 5, 500 μg total lysate. Protein amounts are confirmed in lanes 4 and 6 for FLAG-EhLimA-overexpressing cells and wild-type cells, respectively, each containing 10 μg of total lysate.