Quantification of import inhibition in Nup358-depleted cells

Control or Nup358-depleted cells were transfected with the corresponding cDNA. The cellular localization of the reporter proteins were determined by fluorescence microscopy. The knockdown efficiency of Nup358 was revealed by immunostaining using an α-Nup358 antibody. For quantification, cells were grouped into three categories:

majority of the reporter protein in the nucleus (N>C), equal distribution between nucleus and cytoplasm (N=C) and mainly cytoplasmic localization (C>N). In general, quantification was performed from at least three independent experiments, each with more than 100 cells showing equal expression levels. Error bars depicted the standard deviation of the mean of three independent experiments. Statistical significance of the data was determined applying a two-tailed and heteroscedastic student´s t-test (White, 1980). All calculations and quantifications were performed using Microsoft Office Excel 2007.

In some cases, the analysis of siRNA-treated and control cells revealed considerable variations in the cargo distribution between single experiments. For this reason or for a better comparison between different transfection conditions, the localization in Nup358 knockdown cells overexpressing Nup358 variants was analyzed in relation to the distribution in control cells. The number of control cells in the category N>C was normalized to 100 %, and the distribution in the siRNA-treated cells was calculated in relation to this.

Data analysis and quantification

Fluorescence intensities were measured using the ROI function in the LSM software to get averaged pixel intensities in the designated area, and the background signal was subtracted prior to further analysis. The ratio of nuclear/cytoplasmic fluorescence was plotted for the different conditions.

Images were processed using the LSM Image Browser, AxioVision Rel. 4.8 LE and Adobe Photoshop 6.0.

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2.4.10 Immunoprecipitations

All buffers for immunoprecipitations contained freshly added 2 mM DTT and 1 µg/ml of each aprotinin, leupeptin and pepstatin. The buffers for the GFP-nanotrap also contained 1 mM PMSF.

Immunoprecipitations using NP40-buffer

Immunoprecipitations (IP) are used to isolate and concentrate particular proteins from cell lysate containing many thousands of different proteins. Usage of a specific antibody against that certain protein or its tag causes precipitations which can be coupled to solid substrate later on. In this work, immunoprecipitations were performed from 2-4 10 cm dish/sample using 1 µl affinity-purified α-HA-, α-importin β or α-Nup358 antibody per dish. In case of HA-Nup358 full-length or its truncation mutants, HeLa P4 cells were transfected following the special treatment with 10 % DMSO. Due to low expression, 4x 10 cm dishes were used for one experiment. 48 hr after transfection, 293T HEK cells were washed twice with ice-cold PBS, and 400 µl cold NP40-buffer supplemented with 2 % (w/v) BSA was added per dish. Cells were harvested and incubated on ice for 15 min. The lysate was cleared by centrifugation at 100,000x g using the TLA 100.3 rotor for 20 min. Before adding the antibody, a small sample, referred to as input, was taken and supplemented with the appropriate amount of 2x SDS loading buffer. The antibody was incubated with the lysate for 3 hr, rotating slowly at 4°C before adding 10-15 μl Protein A agarose (Roche) per reaction. For the α-Nup358 antibody, protein G agarose was used. The beads had been equilibrated for at least for 3 hr with NP40-buffer containing 10 % (w/v) BSA. All centrifugation steps with the agarose were performed at 1,000 rpm at 4°C. The samples were then rotated at 4 °C for another 1.5 hr.

The immunoprecipitates were washed 3 times with NP40-buffer. Bound proteins were eluted by either adding 50 µl 2x SDS loading buffer or by a two-step elution with the HA-peptide in case of the α-HA-antibody.

To this end, synthetic HA-peptide [10 mg/ml] was diluted in NP40-buffer to 400 µg/ml, 30 µl were added to the agarose and incubated for 10 min at 30°C by gentle shaking.

After centrifugation, the supernatant was removed and kept on ice, and another round of

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elution followed. Afterwards, the complete eluate was supplemented with the appropriate amount of 4x SDS sample buffer, and subjected to SDS-PAGE followed by western blotting to check the efficiency of the precipitation and to look for interacting proteins.

Immunoprecipitations of GFP-tagged proteins using the GFP-nanotrap

Green fluorescent proteins (GFP) and their variants are widely used to study protein localization and dynamics. For biochemical analysis these GFP fusion proteins and their interacting factors can be isolated efficiently via a special immunoprecipitation where a small GFP binding protein of 13 kDa derived from a llama single chain antibody is coupled to NHS sepharose (Rothbauer et al., 2008).

Transfected 293T HEK cells were lysed in 200 µl ice-cold lysis buffer per 10 cm dish and incubated on ice for 10 min. The cell lysate was then centrifuged for 20 min at 13,000 rpm at 4°C. The supernatant was collected carefully, and its volume was adjusted to 500 µl with ice-cold dilution buffer. A small sample was taken referred to as input. In between, 15 µl beads per sample, were equilibrated with dilution buffer by 2 washing steps and centrifugation at 2,700x g. Cell lysate was added to the sepharose and incubated for 2-3 hr at 4°C while rotating. Afterwards, the beads were washed 3 times with ice-cold wash buffer. In case a binding-assay with recombinant import receptors followed, 2 more washing steps with buffer B, containing 2 % (w/v) BSA, followed. For that, the immunoprecipitated GFP-protein coupled to the beads was transferred to a 500 µl eppendorf tube and progressed as described for in vitro binding assays.

In case no binding-assay was performed, the beads were resuspended in 50 µl 2x SDS sample buffer and boiled at 95°C.

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3 RESULTS

3.1 Nup358 depletion affects nuclear import and not export of