Figure 4.

CSN regulates CRL assembly. (A) Cells were transiently transfected with CSN2^siRNA1 to functionally disrupt the CSN. At 48 h after transfection, mock-transfected and CSN2-knockdown cells were stimulated for different periods of time with TNFα or left untreated, as indicated. The cells were harvested in hypotonic lysis buffer followed by cytosolic/nuclear fractionation. Endogenous β-TrCP was immunoprecipitated from the cytosolic fractions and the immunoprecipitates analysed by SDS–PAGE and Western blotting. Cul1 associated with β-TrCP as well as neddylated Cul1 (Cul1-Nedd8) was immunodetected, using Cul1- or Nedd8-specific antibodies, as indicated. Reduced expression of immunodetected β-TrCP and Cul1 in lysates of CSN2^siRNA1-treated cells was shown by use of protein-specific antibodies. IκBα and phosphorylated IκBα were immunodetected using specific antibodies. Immunodetection of CSN2 was shown as a control for efficient knockdown. Equal amounts of protein in the lysates were controlled by immunodetection of ERK (indicated panels). (B) Endogenous Nedd8 was immunoprecipitated from cytosolic fractions of HeLa cells, transfected with CSN2^siRNA1 or a nontargeting siRNA. Immunoprecipitates from mock-transfected and CSN2^siRNA1-treated cells were analysed by SDS–PAGE and Western blotting. Associated β-TrCP as well as Cul1 was detected using β-TrCP- and Cul1-specific antibodies, respectively. The neddylation state of Cul1 was immunodetected in the total cell lysate using a Cul1-specific antibody. To verify the functional impairment of CSN, hyperneddylation and a decrease in Cul1 expression, as well as increased expression of IκBα in CSN2-knockdown versus mock-transfected cells was immunodetected in the cell lysates using Cul1- or IκBα-specific antibodies, as indicated. ERK was immunodetected to show equal amounts of protein in the cell lysates.