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Deregulation of canonical Wnt signalling and proteasome activity following loss or mutation of MKS1.(a) Immunoblots for total soluble β-catenin, phospho-β-catenin, cyclin D1 and β-actin (loading control) in either wild-type normal or MKS1-mutated immortalised human fibroblasts from an MKS patient (MKS-562) following treatment with MG-132 proteasome inhibitor (+) or vehicle control (-). (b) SUPER-TOPFlash assays of canonical Wnt signalling activity in human MKS1-mutated fibroblasts compared to wild-type control fibroblasts following treatment with control conditioned medium, Wnt5a, Wnt3a, or a mixture of Wnt3a and Wnt5a media, as indicated. Statistical significance of pairwise comparisons is shown (* indicates p < 0.05, paired two-tailed Student t-test). Error bars indicate s.e.m. with results shown for four independent biological replicates. (c) Proteasome activity assays for wild-type or MKS1-mutated human fibroblasts or an irrelevant control (ASPM-mutant fibroblasts), following treatment with c-lactacystin-β-lactone (+) or vehicle control (-). Statistical significance of pairwise comparison as for (b); *** indicates p < 0.001 for three independent biological replicates. Immunoblots show levels of the 20 S proteasome α7 subunit compared to β-actin loading control. (d) Protease activity assays of crude proteasome preparations from Mks1+/+ or Mks1-/- mouse embryonic fibroblasts (MEFs), expressed as pmol AMC released per µg proteasome per hr. Treatment with lactacystin is the assay control. Statistical analysis as for (b); ** indicates p T]+[IVS15-7_35del29] causing the predicted nonsense and splice-site mutations [p.R158*]+[p.P470fs*562]. Additional smaller PCR products in MKS1 patient corresponds to skipping of exon 5 and exon 16, confirmed by Sanger sequencing, due to the frameshift mutation affecting splicing. (b) Immunoblot showing loss of MKS1 protein in MKS1-mutated patient fibroblasts compared to healthy controls; loading control is β-actin. (c) IF microscopy images of wild-type control and MKS1-mutated fibroblasts showing loss of cilia and disorganisation of cytoskeleton in patient cells. Bar graphs quantify reductions in incidence and length of cilia in patient cells. Statistical significance of pairwise comparisons with control for three independent biological replicates are shown (*** p < 0.001, **** p < 0.0001; paired two-tailed Student t-test; error bars indicate s.e.m.) (d) IF microscopy images showing loss of MKS1 ciliary localisation in MKS1-mutated patient fibroblasts compared to wild-type control fibroblasts (indicated by arrowheads). (e) Western blot showing increased levels of polyubiquitinated protein in MKS1-mutated fibroblasts after MG-132 proteasome inhibition. and shUbe2e1 after MG-132 treatment.Figure 1—figure supplement 1—source data 1.Characterisation of MKS1-mutated human patient fibroblasts: full western blots & gels.Characterisation of MKS1-mutated human patient fibroblasts: full western blots & gels.In vivo loss of MKS1 causes deregulated ubiquitin-proteasome processing.Proteasome defects (GFP; green) in Mks1-/- x UbG76V-GFP E12.5 embryonic heart (top panels) and liver (middle panels) as indicated, with accumulation of active β-catenin (red) and GFP-tagged ubiquitin (green) in the neuroepithelium of E12.5 Mks1-/-xUbG76VGFP embryonic neocortex. Scale bars = 50 μm. White frames indicate magnified regions displayed in insets.
Image collected and cropped by CiteAb under a CC-BY license from the following publication: Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1. Elife (2022)
Differential centrifugation of PBMC lysate yields fractions that are highly enriched in endolysosomes. (A) Enrichment of endolysosomes in fractions recovered after differential centrifugation was assessed by western blot. PBMC lysate from which endolysosomes were extracted (lane 1), fraction enriched in endolysosomal proteins LAMP1 and Cathepsin S (lane 2). (B) Isolated endolysosomal (light grey) and cytosol depleted of lysosomal (dark grey) fractions were assayed for activity of cathepsins, aminopeptidases and chymotryptic activity of proteasome in the presence of inhibitors Epoxomicin (Epox) for proteasome, Bestatin (Best) for aminopeptidase, E64 for cathepsins and EDTA as a metal chelator. Mean and SD of replicates from two different donors are plotted. (C) Acid phosphatase assay was performed to confirm enrichment of endolysosomes in different fractions. Figure representative of acid phosphatase activities in one fractionation experiment done in triplicates. Mean and SD are plotted. (D) Two HIV Gag peptides, B57KF11 (KAFSPEVIPMF) and B27KK10 (KRWIILGLNK) were subjected to degradation by equivalent amounts of purified cytosol (blue) or endolysosomes (red). An aliquot of the reaction mixture was stopped at periodic intervals and the percentage of original peptide remaining was determined by HPLC. The half-life of the peptide in both conditions was calculated (36 min in cytosol and 8 min in endolysosomes for B57KF11 and more than 60 min in cytosol and 38 min in endolysosome for B27KK10). Figure 1D is representative of two replicates.
Image collected and cropped by CiteAb under a CC-BY license from the following publication: A simple methodology to assess endolysosomal protease activity involved in antigen processing in human primary cells. BMC Cell Biol (2013)
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