Journal: Kidney360

Article Title: Dynll1-PI31 Interaction Enhances Proteolysis Through the Proteasome, Representing a Novel Therapeutic Target for INF2-Related FSGS

doi: 10.34067/KID.0000000659

Figure Lengend Snippet: Antibodies and assay kits

Article Snippet: Rabbit anti-Dynll1 , Thermo Fisher , PA5-97920.

Techniques: Membrane, Staining, Activity Assay

Journal: Kidney360

Article Title: Dynll1-PI31 Interaction Enhances Proteolysis Through the Proteasome, Representing a Novel Therapeutic Target for INF2-Related FSGS

doi: 10.34067/KID.0000000659

Figure Lengend Snippet: Mouse siRNA duplex sequences

Article Snippet: Rabbit anti-Dynll1 , Thermo Fisher , PA5-97920.

Techniques: Control

Journal: Kidney360

Article Title: Dynll1-PI31 Interaction Enhances Proteolysis Through the Proteasome, Representing a Novel Therapeutic Target for INF2-Related FSGS

doi: 10.34067/KID.0000000659

Figure Lengend Snippet: INF2 R218Q promotes proteasome-mediated degradation of nephrin through Dynll1-PI31 interaction . (A) Increased endogenous Dynll1-PI31 interaction and corresponding reduced Dynll1-INF2 interaction in glomerular lysates of R218Q transgenic mice ( wt/ki and ki/ki versus wt/wt ), as measured by Co-IP and quantified as the fraction of INF2 or PI31 protein in the Dynll1-pulldown relative to the total amount in the lysates. n =3, * P < 0.05 versus wt/wt . (B and C) The degradation of nephrin protein in CHX-treated wt and R218Q KI (R218Q) podocytes with siRNA-mediated knockdown of Dynll1 or PI31, compared with cells transfected with control siRNA (B), or in cells treated with Ciliobrevin D (50 µ M) or bortezomib (100 nM), compared with cells treated with vehicle (0.3% DMSO; C). After normalizing nephrin to β -actin, the percent degradation of nephrin was calculated as ([CHX 0 -CHX 2h ]/CHX 0 )×100%. n =3, * P < 0.05 versus wt+control siRNA, ^ P < 0.05 versus R218Q+control siRNA . # P < 0.05 versus R218Q+Ciliobrevin D . (D) Immunofluorescent staining of nephrin (red) and PI31 (green) in wt or R218Q podocytes with different treatments. The MFI of nephrin staining per podocyte and the percentage of peripheral membrane that was nephrin-positive (perimeter labeled in blue by CellBrite 650 membrane stain, Supplemental Figure 2B ) were quantified for comparison. n =5 cells with well-spread peripheral membrane per assay×three independent assays=15), * P < 0.05 versus wt+control siRNA or wt+DMSO, ^ P < 0.05 versus R218Q+control siRNA or R218Q+DMSO . (E) Increased total and K48-specific polyubiquitinated proteins in cells with bortezomib treatment shown in immunoblots of ubiquitinated proteins in both cell lysates and GST-S5a UIM-pulldowns. Cell lysates incubated with GST (instead of GST- S5a) served as a control. (F) Percentage of proteasome activity in cells with siRNA-mediated PI31 knockdown (normalized to the mean of cells treated with control siRNA) and in cells pretreated with bortezomib (normalized to the mean of cells treated with DMSO control). CHX, cycloheximide; Co-IP, co-immunoprecipitation; DMSO, dimethyl sulfoxide; Dynll1, dynein light chain 1; GST, glutathione-S-transferase; INF2, inverted formin 2; IP, immunoprecipitation; KI, knockin; MFI, mean fluorescent intensity; PI31, proteasomal inhibitor of 31kD; UIM, ubiquitin-interacting motif; wt, wild-type.

Article Snippet: Rabbit anti-Dynll1 , Thermo Fisher , PA5-97920.

Techniques: Transgenic Assay, Co-Immunoprecipitation Assay, Knockdown, Transfection, Control, Staining, Membrane, Labeling, Comparison, Western Blot, Incubation, Activity Assay, Immunoprecipitation, Knock-In

Journal: Kidney360

Article Title: Dynll1-PI31 Interaction Enhances Proteolysis Through the Proteasome, Representing a Novel Therapeutic Target for INF2-Related FSGS

doi: 10.34067/KID.0000000659

Figure Lengend Snippet: PA-activated proteasomes deplete nephrin in R218Q KI mice. (A) Reduced nephrin protein in PAN of R218Q KI mice ( wt/ki and ki/ki versus wt/wt ) shown by coimmunofluorescent staining of nephrin (red) and the podocyte marker, WT1 (green). The autofluorescent background was kept in blue channel to show the outline of the mouse kidney histology. (B) Representative Western blots showing the expression of PI31, INF2, Dynll1 and the SD proteins, nephrin and synaptopodin, in mouse glomerular lysates. (C) β -actin-normalized nephrin protein in glomerular lysates was compared in mice of different genotypes and treatments ( n =6 mice per group). (D) Proteasome activity was measured in the glomerular lysates and compared among mice with different genotypes and treatments ( n =6). The inhibitory effect of 25 µ M Lactacystin added into the lysates demonstrated the successful performance of the assay (proteasome activity [+Lactacystin/−Lactacystin]×100%). (E) Coimmunofluorescent staining of nephrin (green) and Dynll1 (red) in kidney sections of control and PA-treated mice of different genotypes ( wt/wt, wt/ki, and ki/ki ). Scale bar: 20 µ m. By using the Coloc2 plugin of Fiji software, Dynll1-nephrin colocalization was quantified as the Manders overlap coefficient and the Pearson correlation coefficient in five randomly picked glomeruli per mouse kidney section and the mean of measurements per mouse kidney section were calculated for comparison. n =6. * P < 0.05 versus control mice of the same genotype , ^ P < 0.05 versus PA-treated mice+NS treatment control of the same genotype. NS, normal saline; PA, puromycin aminonucleoside; PAN, puromycin aminonucleoside nephropathy; RFU, relative fluorescent units; SD, slit diaphragm; WT1, Wilms tumor 1.

Article Snippet: Rabbit anti-Dynll1 , Thermo Fisher , PA5-97920.

Techniques: Staining, Marker, Western Blot, Expressing, Activity Assay, Control, Software, Comparison, Saline, Wilms Tumor Assay

Journal: Kidney360

Article Title: Dynll1-PI31 Interaction Enhances Proteolysis Through the Proteasome, Representing a Novel Therapeutic Target for INF2-Related FSGS

doi: 10.34067/KID.0000000659

Figure Lengend Snippet: Schematics. In R218Q KI podocytes, Dynll1 is not sequestrated by the R218Q variant of INF2, enabling the capture of Dynll1 by PI31, promoting the coupling of the dynein cargo (nephrin) with the proteasome. When the proteasomal proteases are activated by PA, more nephrin is degraded, decreasing the pool of nephrin available for surface trafficking. This pathogenic process can be broken by PIs. In wt podocytes, Dynll1 is sequestered by INF2 and the proteasome-mediated degradation of nephrin is limited, allowing adequate flow of nephrin for functional targeting to the cell surface and subsequent incorporation into SDs. PIs, proteasome inhibitors.

Article Snippet: Rabbit anti-Dynll1 , Thermo Fisher , PA5-97920.

Techniques: Variant Assay, Functional Assay

Journal: Frontiers in Dementia

Article Title: Quantitative proteomic analysis using a mouse model of Lewy body dementia induced by α-synuclein preformed fibrils injection

doi: 10.3389/frdem.2024.1477986

Figure Lengend Snippet: Protein quantification using TMT-labeled peptides. (A) Fold changes of 660 commonly identified proteins after normalization using peptides with expression of mass reporter (126 Da). (B) Western blot validation of significantly up- and down-regulated proteins including Tubulin polymerization-promoting protein family member 3 (TPPP3), Ras-related protein Rab-10 (RAB10), Calcium/calmodulin-dependent protein kinase type II subunit alpha (CAMK2A), Dynein light chain 1 (DYNLL1). (C) Quantification graphs of expression ratio obtained from triplicate samples and normalized using expression of β-Actin; two-tailed unpaired Student's t -test. * p < 0.05, ** p < 0.01, n.s., not significant from Student's t -test.

Article Snippet: The membrane were incubated with PBS-T (0.05 % Tween 20, v/v) containing bovine serum albumin (5%, w/v) for blocking, and the membranes were applied with antibodies; polyclonal rabbit anti-TPPP3 (15057-1-AP, ProteinTech), polyclonal rabbit anti-RAB10 (11808-1-AP, ProteinTech), polyclonal rabbit anti-CaMKII-α (3357, Cell Signaling Technology), polyclonal rabbit anti-DYNLL1 (A53885, EpigenTek), and rabbit anti-beta-Actin HRP conjugate (13E5, Cell Signaling Technology).

Techniques: Labeling, Expressing, Western Blot, Two Tailed Test