Fig. 3

Znf179 enhances 26S proteasome activity by regulating the protein expression levels of 19S and 20S proteasome subunits. a 293 T cells were co-transfected with Flag-tagged mZnf179 and Ub^G76V-GFP for 24 h and treated with 10 μM MG132 for 6 h as positive control. The cells were stained with anti-Znf179 antibody (red) and the fluorescence intensity of Ub^G76V-GFP (green) was quantified by the ImageXpress® XL System. b The quantified data showed the fluorescence intensities of Ub^G76V-GFP-positive cells in Flag-mZnf179-transfected cells or MG132-treated cells. Data were presented as Mean ± SEM of at least three independent experiments (* p < 0.05, groups were compared by t-test, two-tailed p values). c and d The protein expression levels of 26S proteasome subunits in hippocampus lysates from wild-type and Znf179 knockout mice were quantified by mass spectrometry analysis. After In-gel trypsin digestion and Zip-tip purification, samples were run on a LTQ-Orbitrap mass spectrometer and analyzed by Mascot 2.3.02 software to identify matched proteins. Further quantification was accomplished with Porgenesis LC-MS software. The differential expressions of peptides related to the 26S proteasome were presented as the ratio in knockout to wild-type. The MS results were normalized by the internal controls, BSA and β-actin, and were further classified by Ingenuity pathway analysis (IPA). Several proteins with significant differences (expression level ≥ 1.3-fold, p < 0.05, peptides ≥2) were associated with ubiquitin-proteasome systems, including the 20S proteasome subunits PSMA2, PSMB1, PSMB3 and PSMB4. e Protein expression patterns of the 19S proteasome subunits, PSMC6 and PSMD10, and 20S proteasome subunits, PSMA1 between wild-type and Znf179-knockout mice brain were analyzed by Western blotting. β-actin was used as an internal control