Figure 2
From: The E3 ubiquitin ligase RNF121 is a positive regulator of NF-κB activation
RNF121 is a Golgi Apparatus-anchored E3 ubiquitin ligase. (A) Scheme showing the main domains of RNF121. The mutations used in Figure 1E and 1F are also indicated. (B) HEK293T cells were transfected with a Myc-tagged plasmid coding for RNF121. 24 hrs later, crude heavy membranes (HM) and cytosolic (Cyt.) fractions were analyzed by immunoblotting as indicated. Calnexin and GAPDH served as purity controls for the HM or cytosolic fraction respectively. (Ub)n indicates poly-ubiquitylated species. (C) Crude heavy membranes (HM) and cytosolic (Cyt.) fractions from HEK293T were analyzed by immunoblotting as indicated. (D) HeLa cells were transfected with a Myc-tagged plasmid coding for RNF121. 24 hrs later, the localization of myc-RNF121 was investigated by immunofluorescence. GM130 was used as a marker for the Golgi Apparatus. Nuclei were stained with DAPI. Representative images are shown, with the boxed areas enlarged on the right. (E) Analysis of the localization of endogenous RNF121 in HeLa cells by immunofluorescence. (F) HeLa were transfected with a control nonspecific siRNA or with a siRNA raised against RNF121. 72 hrs later the Golgi Apparatus morphology was examined. As a control, HeLa cells were treated with Brefeldin A (Bref. A) (50 ng/ml) for 3 hrs. Scale bar : 20 μm.