Journal: Kidney360

Article Title: Dynein-Mediated Trafficking: A New Mechanism of Diabetic Podocytopathy

doi: 10.34067/KID.0006852022

Figure Lengend Snippet: Antibodies and fluorescent probes (immunofluorescent, Western blotting, immunoprecipitation]

Article Snippet: Table 2 Antibody Dilution Company Cat# Mouse antinephrin (G-8) IF: 1:100 Santa Cruz sc-376522 Mouse antinephrin conjugated to agarose IP: 2 μg/400 μl lysate Santa Cruz sc-376522 AC Normal mouse IgG conjugated to agarose IP: 2 μg/400 μl lysate Santa Cruz sc-2343 Rabbit antinephrin WB: 1:1000 Invitrogen PA5-91907 Sheep anti-DCTN1 WB: 1:1000 IF: 1:100 R&D Systems AF6657 Mouse anti-Dynll1 IF: 1:100 Santa Cruz sc-136287 Rabbit anti-Dynll1 WB: 1:1000 IF: 1:100 Thermo Fisher PA5-97920 Rabbit anti-INF2 IF: 1:100 Bethyl Lab A303-427A Rabbit anti-HDAC6 WB: 1:1000 Novus NBP1-78981 Donkey anti-mouse (Alexa Fluor 488) IF: 1:200 Thermo Fisher A32766 Donkey anti-rabbit (Alexa Fluor 488) IF: 1:200 Thermo Fisher A32790 Donkey anti-mouse (Alexa Fluor 594) IF: 1:200 Thermo Fisher A32744 Donkey anti-sheep (Alexa Fluor 488) IF: 1:200 Thermo Fisher A-11015 Mouse anti-rabbit IgG-HRP WB: 1:5000 Santa Cruz sc-2357 Rabbit anti-sheep IgG-HRP WB: 1:5000 Thermo Fisher 31480 Biotin mouse anti-ubiquitin WB: 1:1000 Invitrogen 13-6078-82 Mouse anti β-actin-HRP WB: 1:5000 Santa Cruz sc-47778 Mouse anti-WT1 IF: 1:100 Novus NBP24460700 Mouse anti-acetylated α-tubulin IF: 1:200 Santa Cruz sc-23950 Acti-stain 488 phalloidin Cytoskeleton PHDG1-A ViaFluor microtubule stains (488) Biotium 70062 Open in a separate window IF, immunofluorescent; IP, immunoprecipitation; WB, Western blotting; INF2, inverted formin 2; HDAC6, histone deacetylase 6; HRP, horseradish peroxidase.

Techniques: Western Blot, Immunoprecipitation

Journal: Kidney360

Article Title: Dynein-Mediated Trafficking: A New Mechanism of Diabetic Podocytopathy

doi: 10.34067/KID.0006852022

Figure Lengend Snippet: Antibodies and fluorescent probes (immunofluorescent, Western blotting, immunoprecipitation]

Article Snippet: Table 2 Antibody Dilution Company Cat# Mouse antinephrin (G-8) IF: 1:100 Santa Cruz sc-376522 Mouse antinephrin conjugated to agarose IP: 2 μg/400 μl lysate Santa Cruz sc-376522 AC Normal mouse IgG conjugated to agarose IP: 2 μg/400 μl lysate Santa Cruz sc-2343 Rabbit antinephrin WB: 1:1000 Invitrogen PA5-91907 Sheep anti-DCTN1 WB: 1:1000 IF: 1:100 R&D Systems AF6657 Mouse anti-Dynll1 IF: 1:100 Santa Cruz sc-136287 Rabbit anti-Dynll1 WB: 1:1000 IF: 1:100 Thermo Fisher PA5-97920 Rabbit anti-INF2 IF: 1:100 Bethyl Lab A303-427A Rabbit anti-HDAC6 WB: 1:1000 Novus NBP1-78981 Donkey anti-mouse (Alexa Fluor 488) IF: 1:200 Thermo Fisher A32766 Donkey anti-rabbit (Alexa Fluor 488) IF: 1:200 Thermo Fisher A32790 Donkey anti-mouse (Alexa Fluor 594) IF: 1:200 Thermo Fisher A32744 Donkey anti-sheep (Alexa Fluor 488) IF: 1:200 Thermo Fisher A-11015 Mouse anti-rabbit IgG-HRP WB: 1:5000 Santa Cruz sc-2357 Rabbit anti-sheep IgG-HRP WB: 1:5000 Thermo Fisher 31480 Biotin mouse anti-ubiquitin WB: 1:1000 Invitrogen 13-6078-82 Mouse anti β-actin-HRP WB: 1:5000 Santa Cruz sc-47778 Mouse anti-WT1 IF: 1:100 Novus NBP24460700 Mouse anti-acetylated α-tubulin IF: 1:200 Santa Cruz sc-23950 Acti-stain 488 phalloidin Cytoskeleton PHDG1-A ViaFluor microtubule stains (488) Biotium 70062 Open in a separate window IF, immunofluorescent; IP, immunoprecipitation; WB, Western blotting; INF2, inverted formin 2; HDAC6, histone deacetylase 6; HRP, horseradish peroxidase.

Techniques: Western Blot, Immunoprecipitation

Journal: Blood Cancer Discovery

Article Title: Mis-splicing of Mitotic Regulators Sensitizes SF3B1-Mutated Human HSCs to CHK1 Inhibition

doi: 10.1158/2643-3230.BCD-23-0230

Figure Lengend Snippet: Pervasive mis-splicing of cell division and genome maintenance genes in CD34 + HSPCs. A, Percent mis-splicing of TMEM14C , MAP3K7 , DYNLL1 , and ORAI2 mRNA in patients with SF3B1 WT or mutant (MUT) MDS, iPSC-derived HSPCs, K562 cells, and edited CD34 + CB and PB HSPCs, or normal BM. Median and range; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-sided Mann–Whitney U test. B, Gene ontology (GO) analysis of a3′ss mis-splicing targets in SF3B1 K700E edited PB CD34 + HSPCs. GO performed using Metascape showing summary terms; ≥10% mis-splicing, Bayes factor ≥5. C, Overlap between mis-spliced a3′ss events in CD34 + CB, CD34 + PB and K562 SF3B1 K700E cells, and list of recurrently mis-spliced genes in all three cell types (right); ≥10% mis-splicing, Bayes factor ≥5. D, STRING protein–protein interaction network of CB/PB a3′ss mis-spliced genes in the cell division GO category; disconnected nodes removed, line thickness proportional to interaction score >0.40. E, GO analysis of genes downregulated in SF3B1 K700E CD34 + CB/PB HSPCs (log 2 fold change ≤ −0.5; P < 0.05). GO performed using Metascape showing summary terms. F, Hallmark pathways upregulated or downregulated in SF3B1 K700E vs. control edited CD34 + CB/PB HSPCs; >25 genes, FDR < 0.01. NES, normalized enrichment score. G, Significantly down- or up-regulated genes among 186 genes with conserved a3′ss mis-splicing in both CB and PB CD34 + HSPCs from Supplementary Fig. S2B. Expression ranked by log 2 fold change in SF3B1 K700E vs. control edited CD34 + HSPCs, P < 0.1. Mitosis-related terms are shown in purple color.

Article Snippet: Lysates were resolved by 4% to 20% SDS-PAGE (Bio-Rad) and immunoblotted with antibodies for GAPDH (1:10,000, Abcam, ab9485), HSP90 (1:10,000, BD Biosciences #610419), RUNX1 (1:1,000, Santa Cruz sc-365644), STAG2 (1:1,000, Santa Cruz sc-81852), BUBR1 (1:1,000, BD Biosciences #612502), CDC27 (1:500, Cell Signaling Technologies #12530), pCHK1 S345 (1:1,000, Cell Signaling Technologies #2348), CHK1 (1:1,000, Cell Signalling Technology #2360), DYNLL1 (1:5,000, abcam #51603).

Techniques: Mutagenesis, Derivative Assay, MANN-WHITNEY, Control, Expressing

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