Fig. 7: UCH-L1 interacts with the proteasome.

a Scheme of the standard proteasome (proteolytic subunits β1c, β2c and β5c) and of the immunoproteasome (proteolytic subunits β1i, β2i and β5i). b, c Experimental MN was induced by i.v. injection of sheep anti-murine podocyte antibodies (AP-abs) in UCH-L1 overexpressing (WT OE and I93M OE, respectively) and their littermate (Ctrl) mice. Kidneys were collected on day 14. b Glomerular immunoblot analyses of main (β5c and β5i) proteasomal subunit abundance normalized to β-actin. Pooled values of 4 independent experiments (WT-UCH-L1, n = 20 Ctrl or n = 9 WT OE) or of 2 independent experiments (I93M-UCH-L1, n = 20 Ctrl or n = 9 I93M OE) for each subunit are shown as mean + /-SEM, ***p = 0.0004 (β5c), ***p = 0.0002 (β5i), two-tailed Mann Whitney U test. c Confocal images showing co-localization of respective proteolytic proteins (green) with UCH-L1 (red) in podocytes (marked by asterisks). Nephrin (blue) was used to depict the glomerular filtration barrier and DNA was stained with Hoechst (grey). Graphs: semiquantitative analysis of spatial proximity of β5c and β5i with UCH-L1 in selected podocytes (yellow overlay in framed area), respectively; strength of positive (0 to +1) or negative (0 to −1) linear relationship represented by Pearson correlation coefficient. The box illustrates the interquartile range (IQR), median line and whiskers including the lowest and highest values within ±1.5 × IQR and above. Values consist of n = 3 (WT-UCH-L1 or I93M-UCH-L1), two-tailed Unpaired t-test. d, e Interaction studies of UCH-L1 with the proteasome by using HEK293T cells transiently transfected with human (hu) wildtype UCH-L1-flag (WT-UCH-L1) and hu-I93M-UCH-L1-flag (I93M-UCH-L1) protein. Representative immunoblot analyses of 1 experiment demonstrate interaction between UCH-L1 and the proteasome. WT-UCH-L1 and non-functional I93M-UCH-L1 protein were co-immunoprecipitated with the 20S proteasome (d, lower panel). Successful proteasome IP is shown via immunoblot to the α6 subunit (d, upper panel). Vice versa the proteasome (detected via the α4 subunit, (e, lower panel)) was co-immunoprecipitated with UCH-L1 proteins, respectively (e, upper panel). Untransfected HEK293T cells served as control. IgG LC = immunoglobulin G light chain of the capture antibody. Source data are provided as a Source Data file.