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. 2006 Sep;26(18):6772–6785. doi: 10.1128/MCB.00342-06

FIG. 1.

FIG. 1.

Efficient and specific depletion of Nup214 and Nup358 by RNA interference. HeLa cells were mock treated or transfected with specific siRNAs against Nup214 or Nup358. (A) Different amounts (100%, 50%, 25%) of cell lysates from mock-treated cells were compared to lysates from nucleoporin-depleted cells by immunoblotting, detecting Nup358, Nup214, and p62. A parallel Coomassie stain served as a loading control. Note the slightly lower protein concentration in the lysate obtained from Nup214-2-transfected cells. (B) Mock- or siRNA-treated cells were analyzed by triple immunofluorescence, using anti-Nup358, anti-Nup214, and the antinucleoporin antibody RL1, as indicated. (C) Mock- or siRNA-treated cells were analyzed by double immunofluorescence for Nup88 and Nup214 or Nup358. Nucleoporin-depleted cells are indicated by arrows. For a better comparison of protein levels, nucleoporin-depleted cells were mixed with untransfected cells. Antibodies used for detection of Nup214 or Nup358 are indicated in the individual pictures (right panel). (D) Different amounts (100%, 50%) of cell lysates from mock-treated cells and Nup214-depleted cells were analyzed by immunoblotting, detecting Nup214 and Nup88. A parallel Coomassie stain served as a loading control. Note that our commercial anti-Nup88 antibody did not allow a reliable quantitative analysis of the codepletion.