Fig. 2.

p62 depletion impairs non-degradative NEMO ubiquitination. (A) Left panel: HEK293 cells were trasfected with lentiviral vectors encoding for 4 different shRNAs targeting human p62 or a control shRNA (scramble). After selection, p62 mRNA levels normalized to actin were quantified by real-time PCR. Right panels: HEK293 cells were transfected with empty vector or with vectors expressing a FLAG-tagged version of p62 or an shRNA targeting human p62, as indicated. 24 h later, cell lysates were separated by SDS-PAGE and blotted onto membranes probed with anti FLAG or anti p62 antibodies, as indicated. (B) Control and p62-depleted HEK293 cells were treated with TNFα (10 ng/ml) for the indicated time periods and the ubiquitination state of NEMO was monitored by immunoblot assay. (C) Control and p62-depleted HEK293 cells were stimulated with TNFα for the indicated time periods and cell lysates were immunoprecipitated with anti-NEMO antibody. The ubiquitination state of NEMO was monitored by immunoblot assay probed with anti-ubiquitin (upper panel) or anti-NEMO (lower panel). (D) Control and p62-depleted HEK293 cells were treated with IL-1β (20 ng/ml) and the ubiquitination state of NEMO was monitored as in C). (E) Control and p62-depleted HEK293 cells were stimulated with TNFα for the indicated time periods and cell lysates were immunoprecipitated with anti-NEMO antibody in denaturing condition (see Section 2). The ubiquitination state of NEMO was monitored by immunoblot assay probed with anti-ubiquitin.