(a) HEK293 cells were transfected with wild type or K472E/R473E mutant Cul1-V5, followed by Western blotting with V5 antibody. (b–d) Lysates from cells transfected with wild type or mutant (K472E/R473E) Cul1-V5 were subjected to immunoprecipitation using V5 antibody followed by Western blotting of lysates and immunoprecipitates with the indicated antibodies. In (d) dnUbc12 tet-on cells were used for transfection and induced with 1 µg/ml tetracycline for 24 hours prior to cell lysis in order to block Cul1 neddylation. (e) Cells were transfected in 60 mm dishes with 1 µg wild type or K472E/R473E mutant Cul1-V5 and 1.5 µg dnCul1-V5 or empty vector, as indicated. After two days, all plates were transfected with 10 µg of recombinant GST-CAND1 in the presence or absence of TurboFect protein transfection reagent. One hour after protein transfection, cells were rinsed and lysed and cell lysates subjected to V5 immunoprecipitation. The immunoprecioitates were analyzed by Western blotting with GST and V5 antibodies. As expected, no GST-CAND1 was observed in cell lysate and V5 immunoprecipitates when no protein transfection agent was included (see lanes 5 and 10). When GST was transfected into cells as a negative control, no binding to Cul1 could be observed (not shown).

(a,b) To compare cellular CAND1 and Cul1 protein expression, transfected and immunoprecipitated Cul1-V5 (lane 1 and CAND1-V5 (lane 2) were used as protein standards. Endogenous amounts of CAND1 and Cul1 protein in total lysate, nuclear and cytoplasmic fractions was measured in Western blots and compared to the respective protein standards by densitometry. Direct comparison of the two protein standards by Western blotting with V5 antibody (lower panel in (a)) allowed for calculation of the ratios of endogenous CAND1 to Cul1. Densitometry measurements from four independent experiments gave the means and S.E.M. values presented in (b). (c) The purity of the nuclear and cytoplasmic fractions, which were prepared as described under Methods, was confirmed by Western blotting with PARP and GAPDH antibodies.

HEK293 cells were cotransfected with the indicated plasmids. Cell lysates were used for immunoprecipitation with FLAG antibody followed by Western blotting.

(a) Western blot analysis of two different clones of HEK293 cells with stable expression of N-terminally FLAG-tagged and C-terminally HA-tagged CAND1 under a tetracycline-inducible promoter. Western blotting with CAND1 antibody indicates that the stably transfected CAND1 is expressed at physiological concentrations. (b) Cells harboring the stably transfected CAND1 were induced with 1 µg/ml of tetracycline for 24 hours. Control HEK 293 cells and tetracycline-induced cells were harvested and lysed with hypotonic lysis buffer consisting of 25 mM Tris, 2 mM EDTA, 2 mM EGTA and complete protease inhibitor cocktail tablet (Roche). Lysates were subjected to immunoprecipitation using anti-FLAG antibody to immunoprecipitate FLAG-CAND1-HA, immunoprecipitates were washed four times with 1X phosphate buffer saline (1X PBS) and analyzed by SDS-PAGE and Coomassie blue staining. A number of bands with a molecular weight of around 85 kDa were detected that were not present in the control. Mass spectrometric analysis of these bands revealed their identity as Cul1 (score:173; 12 detected peptides), Cul2 (score: 200; 17 detected peptides), Cul3 (score:63; 2 detected peptides), and Cul5 (score:47; 2 detected peptides). (c) HEK293 cells were transfected in 60-mm cell culture plates for two days with V5-tagged expression constructs for the cullin homologs indicated at the top of each panel. The cells were lysed, and the lysates were subjected to V5 immunoprecipitation, as described under Methods. Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with the indicated antibodies.

Cells were transfected with siRNA targeting CAND1 or CSN5 for three days, as described under Methods. In lanes 2 to 4, 20 nM of individual siRNA was transfected. In lane 5, 10 nM of each CAND1 and CSN5 siRNA and in lane 6, 20 nM of both siRNAs was used. Cul1 neddylation was assessed by Western blotting with Cul1 antibody. Densitometry analysis of the ratio of neddylated to unneddylated Cul1 is presented in the lower panel which is derived from two independent experiments which showed very similar trends.

(a) In the left panel, untransfected HEK293 cells were fractionated into nuclear and cytoplasmic fractions as described under Methods. Equal amounts of total, nuclear and cytoplasmic proteins were loaded onto SDS gels and analyzed by Western blotting using CAND1 and Cul1 antibodies. The results shown are representative of three independent experiments. To quantify the ratio of nuclear versus cytoplasmic proteins, densitometry analysis was carried out and the results are mentioned in the text. In the right panel, cells were transiently transfected with FLAG-CAND1-HA, followed by preparation of nuclear and cytoplasmic fractions and Western blotting using the indicated antibodies. (b) Immunofluorescence staining of transfected HEK293 cells was carried out using FLAG antibody, as described under Methods.

(a) dnCul1 tet-on cells were transfected with full length Cul1-FLAG as indicated and dnCul1-V5 expression was induced by adding 1 µg/ml tetracycline during the last 24 hours before cell lysis. Cell lysates were subjected to FLAG immunoprecipitation and Western blotting with the indicated antibodies. (b) Cells were transfected with full length Cul1-V5 or dnCul1-V5, as indicated, followed by V5 immunoprecipitation and Western blotting of immunoprecipitates and cell lysates with CAND1 and V5 antibodies. (c) Cells were transfected for three days with negative control or Skp1 siRNA (20 nM) and for the last two days with Cul1-FLAG plasmid as indicated. FLAG immunoprecpitation was then carried out followed by Western blotting with the indicated antibodies. The band labeled with NS in the CAND1 blot corresponds to a non-specific band.