sod1 24 Search Results


mouse anti sod1  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology mouse anti sod1
Mouse Anti Sod1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sod1+24/pmc09187739-195-19-23?v=Santa+Cruz+Biotechnology
Average 96 stars, based on 1 article reviews
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gene exp adora2a rn00583935 m1  (Thermo Fisher)
93
Thermo Fisher gene exp adora2a rn00583935 m1
Gene Exp Adora2a Rn00583935 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sod1+24/pm29718705-150-13--1?v=Thermo+Fisher
Average 93 stars, based on 1 article reviews
gene exp adora2a rn00583935 m1 - by Bioz Stars, 2026-08
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anti sod1  (Santa Cruz Biotechnology)
95
Santa Cruz Biotechnology anti sod1
Anti Sod1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sod1+24/pm20127037-58-18-20?v=Santa+Cruz+Biotechnology
Average 95 stars, based on 1 article reviews
anti sod1 - by Bioz Stars, 2026-08
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gene exp 18s hs03003631 g1  (Thermo Fisher)
90
Thermo Fisher gene exp 18s hs03003631 g1
Gene Exp 18s Hs03003631 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sod1+24/pmc06726894__EMBR___20___e48084___s003-0-8--1?v=Thermo+Fisher
Average 90 stars, based on 1 article reviews
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2011 rb sod1  (Proteintech)
96
Proteintech 2011 rb sod1
Figure 1. Patient neurons exhibit high levels of disordered soluble and insoluble <t>SOD1</t> protein without impact on the major clearance pathways (A) Experimental schematic of MN generation and biochemical fractionation for ELISA and western blot (WB) analysis of SOD1 protein. (B) Representative images of iPSC-differentiated MNs expressing ISL1/2 and TUJ1. Scale bar, 20 mm. (C) Quantification of soluble disordered SOD1 by ELISA (n = 5 independent biological replicates, two-way ANOVA across time and genotypes, p = 0.4254; Sidak’s multiple comparisons test per time point, day 16 **p = 0.0068, day 30 **p = 0.0029, day 35 *p = 0.0215, day 51 ***p = 0.0003). (D and E) WB analysis (D) and quantification (E) of detergent-insoluble SOD1 (n = 6 independent biological replicates, two-way ANOVA across time and ge- notypes, *p = 0.046; Sidak’s multiple comparisons test per time point, day 16 p = 0.8352, day 25 *p = 0.0120, day 30 ***p = 0.0005, day 35 ****p < 0.0001, day 40 ****p < 0.0001, day 51 **p = 0.0022). The detergent-insoluble SOD1 is expressed relative to the normalized soluble SOD1. (F) Assessment of the ubiquitination flux in mutSOD1 and isogenic control MNs by WB. The proteasome activity was blocked with MG132 (10 mM, 8 h), and polyubiquitinated proteins were isolated from total cell lysate extracts using TUBE magnetic beads. Alternatively, the autophagosome-lysosome fusion was blocked with the inhibitor bafilomycin A1 (20 nM, 24 h), and the polyubiquitinated proteins were isolated in the same way. Representative blots (top) and quantification (bottom). Unpaired t test, for MG132 treatment, n = 6 independent biological replicates, p = 0.236; for BAFA1 treatment, n = 3 independent biological replicates, p = 0.240; ns, not significant.
2011 Rb Sod1, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sod1+24/pm37776851-197-62-83?v=Proteintech
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2011 rb sod1 - by Bioz Stars, 2026-08
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sod1 activity  (Novus Biologicals)
94
Novus Biologicals sod1 activity
Figure 1. Patient neurons exhibit high levels of disordered soluble and insoluble <t>SOD1</t> protein without impact on the major clearance pathways (A) Experimental schematic of MN generation and biochemical fractionation for ELISA and western blot (WB) analysis of SOD1 protein. (B) Representative images of iPSC-differentiated MNs expressing ISL1/2 and TUJ1. Scale bar, 20 mm. (C) Quantification of soluble disordered SOD1 by ELISA (n = 5 independent biological replicates, two-way ANOVA across time and genotypes, p = 0.4254; Sidak’s multiple comparisons test per time point, day 16 **p = 0.0068, day 30 **p = 0.0029, day 35 *p = 0.0215, day 51 ***p = 0.0003). (D and E) WB analysis (D) and quantification (E) of detergent-insoluble SOD1 (n = 6 independent biological replicates, two-way ANOVA across time and ge- notypes, *p = 0.046; Sidak’s multiple comparisons test per time point, day 16 p = 0.8352, day 25 *p = 0.0120, day 30 ***p = 0.0005, day 35 ****p < 0.0001, day 40 ****p < 0.0001, day 51 **p = 0.0022). The detergent-insoluble SOD1 is expressed relative to the normalized soluble SOD1. (F) Assessment of the ubiquitination flux in mutSOD1 and isogenic control MNs by WB. The proteasome activity was blocked with MG132 (10 mM, 8 h), and polyubiquitinated proteins were isolated from total cell lysate extracts using TUBE magnetic beads. Alternatively, the autophagosome-lysosome fusion was blocked with the inhibitor bafilomycin A1 (20 nM, 24 h), and the polyubiquitinated proteins were isolated in the same way. Representative blots (top) and quantification (bottom). Unpaired t test, for MG132 treatment, n = 6 independent biological replicates, p = 0.236; for BAFA1 treatment, n = 3 independent biological replicates, p = 0.240; ns, not significant.
Sod1 Activity, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sod1+24/pm41615536-64-16-18?v=Novus+Biologicals
Average 94 stars, based on 1 article reviews
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sod1/cu-zn sod activity assay kit (colorimetric)  (Novus Biologicals)
N/A
SOD1/Cu-Zn SOD Activity Assay Kit (Colorimetric)
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sod1/cu-zn sod antibody [alexa fluor® 350]  (Novus Biologicals)
N/A
The SOD1/Cu-Zn SOD Antibody [Alexa Fluor® 350] from Novus is a SOD1/Cu-Zn SOD antibody to SOD1/Cu-Zn SOD. This antibody reacts with Human, Mouse, Rat, Bovine. The SOD1/Cu-Zn SOD antibody has been validated for the following
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sod1/cu-zn sod antibody [alexa fluor® 647]  (Novus Biologicals)
N/A
The SOD1/Cu-Zn SOD Antibody [Alexa Fluor® 647] from Novus is a SOD1/Cu-Zn SOD antibody to SOD1/Cu-Zn SOD. This antibody reacts with Human, Mouse, Rat, Bovine. The SOD1/Cu-Zn SOD antibody has been validated for the following
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sod1/cu-zn sod antibody [dylight 594]  (Novus Biologicals)
N/A
The SOD1/Cu-Zn SOD Antibody [DyLight 594] from Novus is a SOD1/Cu-Zn SOD antibody to SOD1/Cu-Zn SOD. This antibody reacts with Human, Mouse, Rat, Bovine. The SOD1/Cu-Zn SOD antibody has been validated for the following applications:
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sod1/cu-zn sod antibody [biotin]  (Novus Biologicals)
N/A
The SOD1/Cu-Zn SOD Antibody [Biotin] from Novus is a SOD1/Cu-Zn SOD antibody to SOD1/Cu-Zn SOD. This antibody reacts with Human, Mouse, Rat, Bovine. The SOD1/Cu-Zn SOD antibody has been validated for the following applications: Western
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sod1/cu-zn sod antibody [dylight 350]  (Novus Biologicals)
N/A
The SOD1/Cu-Zn SOD Antibody [DyLight 350] from Novus is a SOD1/Cu-Zn SOD antibody to SOD1/Cu-Zn SOD. This antibody reacts with Human, Mouse, Rat, Bovine. The SOD1/Cu-Zn SOD antibody has been validated for the following applications:
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Image Search Results


Figure 1. Patient neurons exhibit high levels of disordered soluble and insoluble SOD1 protein without impact on the major clearance pathways (A) Experimental schematic of MN generation and biochemical fractionation for ELISA and western blot (WB) analysis of SOD1 protein. (B) Representative images of iPSC-differentiated MNs expressing ISL1/2 and TUJ1. Scale bar, 20 mm. (C) Quantification of soluble disordered SOD1 by ELISA (n = 5 independent biological replicates, two-way ANOVA across time and genotypes, p = 0.4254; Sidak’s multiple comparisons test per time point, day 16 **p = 0.0068, day 30 **p = 0.0029, day 35 *p = 0.0215, day 51 ***p = 0.0003). (D and E) WB analysis (D) and quantification (E) of detergent-insoluble SOD1 (n = 6 independent biological replicates, two-way ANOVA across time and ge- notypes, *p = 0.046; Sidak’s multiple comparisons test per time point, day 16 p = 0.8352, day 25 *p = 0.0120, day 30 ***p = 0.0005, day 35 ****p < 0.0001, day 40 ****p < 0.0001, day 51 **p = 0.0022). The detergent-insoluble SOD1 is expressed relative to the normalized soluble SOD1. (F) Assessment of the ubiquitination flux in mutSOD1 and isogenic control MNs by WB. The proteasome activity was blocked with MG132 (10 mM, 8 h), and polyubiquitinated proteins were isolated from total cell lysate extracts using TUBE magnetic beads. Alternatively, the autophagosome-lysosome fusion was blocked with the inhibitor bafilomycin A1 (20 nM, 24 h), and the polyubiquitinated proteins were isolated in the same way. Representative blots (top) and quantification (bottom). Unpaired t test, for MG132 treatment, n = 6 independent biological replicates, p = 0.236; for BAFA1 treatment, n = 3 independent biological replicates, p = 0.240; ns, not significant.

Journal: Cell reports

Article Title: Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.

doi: 10.1016/j.celrep.2023.113160

Figure Lengend Snippet: Figure 1. Patient neurons exhibit high levels of disordered soluble and insoluble SOD1 protein without impact on the major clearance pathways (A) Experimental schematic of MN generation and biochemical fractionation for ELISA and western blot (WB) analysis of SOD1 protein. (B) Representative images of iPSC-differentiated MNs expressing ISL1/2 and TUJ1. Scale bar, 20 mm. (C) Quantification of soluble disordered SOD1 by ELISA (n = 5 independent biological replicates, two-way ANOVA across time and genotypes, p = 0.4254; Sidak’s multiple comparisons test per time point, day 16 **p = 0.0068, day 30 **p = 0.0029, day 35 *p = 0.0215, day 51 ***p = 0.0003). (D and E) WB analysis (D) and quantification (E) of detergent-insoluble SOD1 (n = 6 independent biological replicates, two-way ANOVA across time and ge- notypes, *p = 0.046; Sidak’s multiple comparisons test per time point, day 16 p = 0.8352, day 25 *p = 0.0120, day 30 ***p = 0.0005, day 35 ****p < 0.0001, day 40 ****p < 0.0001, day 51 **p = 0.0022). The detergent-insoluble SOD1 is expressed relative to the normalized soluble SOD1. (F) Assessment of the ubiquitination flux in mutSOD1 and isogenic control MNs by WB. The proteasome activity was blocked with MG132 (10 mM, 8 h), and polyubiquitinated proteins were isolated from total cell lysate extracts using TUBE magnetic beads. Alternatively, the autophagosome-lysosome fusion was blocked with the inhibitor bafilomycin A1 (20 nM, 24 h), and the polyubiquitinated proteins were isolated in the same way. Representative blots (top) and quantification (bottom). Unpaired t test, for MG132 treatment, n = 6 independent biological replicates, p = 0.236; for BAFA1 treatment, n = 3 independent biological replicates, p = 0.240; ns, not significant.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-VCP mouse primary antibody Genetex GTX101089; RRID:AB_1952544 Anti-SOD1 rabbit primary antibody Abcam ab51254; RRID:AB_882757 Anti-MAP2 chicken primary antibody Abcam ab5392; RRID:AB_2138153 Anti-b-Tubulin III rabbit primary antibody Millipore-Sigma T2200-200UL; RRID:AB_262133 Anti-Choline Acetyltransferase goat primary antibody Millipore-Sigma AB144P-200UL; RRID:AB_2079751 Anti-ISL1/2 mouse primary antibody DSHB (University of Iowa) 39.4D5; RRID:AB_2314683 ms SOD1 134.2 (57–72) Umeå University Zetterstrom et al., 2011 rb SOD1 (24–39) Umeå University Zetterstrom et al., 2011 VCP Abcam ab109240; RRID:AB_10862588 Ub (P4D1) Santa-Cruz Biotechnology sc-8017; RRID:AB_628423 p62/SQSTM1 Proteintech 18420-1-AP; RRID:AB_10694431 HSPB1 Proteintech 18284-1-AP; RRID:AB_2295540 Normal Rabbit IgG Cell Signaling 2729S; RRID:AB_1031062 Myc-Tag (9B11) Cell Signaling 2276S; RRID:AB_331783 Alexa Fluor 488 goat anti-mouse secondary antibody Jackson Immuno Research (Fisher Scientific) A11001; RRID:AB_2534069 Alexa Fluor 594 donkey anti-rabbit secondary antibody Jackson Immuno Research (Fisher Scientific) A21207; RRID:AB_141637 Alexa Fluor 647 donkey anti-chicken secondary antibody Jackson Immuno Research (Fisher Scientific) 703-606-155; RRID:AB_2340380 Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 Invitrogen A21202; RRID:AB_141607 Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 Invitrogen A11055; RRID:AB_2534102 Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 647 Invitrogen A31573; RRID:AB_2536183 DAPI Invitrogen H21492 Cell Culture Reagents mTeSR Stem Cell Technologies 5820 N-2 Supplement (100X) Thermo Fisher Scientific 17502001 B-27 Supplement (50X), serum free Thermo Fisher Scientific 17504001 GlutaMAX Supplement Thermo Fisher Scientific 35050061 MEM Non-Essential Amino Acids Solution (100X) Thermo Fisher Scientific 11140050 DMEM/F-12 Thermo Fisher Scientific 11320082 Neurobasal Medium Thermo Fisher Scientific 21103049 EdU Thermo Fisher Scientific A10044 Trypsin-EDTA (0.25%), phenol red Thermo Fisher Scientific 25200072 TrypLETM Express Enzyme (1X), no phenol red Thermo Fisher Scientific 12604013 1X PBS Corning MT21040CV Ultrapure water with 0.1% Gelatin Millipore ES006B Accutase Innovative Cell Technologies AT 104-500 SB431542 DNSK International DNSK-KI-12 SAG DNSK International DNSK-SMO-1 SU5402 DNSK International DNSK-KI-11 (Continued on next page) 18 Cell Reports 42, 113160, October 31, 2023

Techniques: Fractionation, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Ubiquitin Proteomics, Control, Activity Assay, Isolation, Magnetic Beads

Figure 2. Mutant SOD1 iPSC-derived patient neurons exhibit slower protein degradation dynamics of a small panel of proteins including VCP/p97 (A) Experimental schematic of SILAC-MS pulse-chase strategy for iPSC-MNs. (B) Assessment of labeling efficiency on day-30 MNs based on the quantification of heavy labeled peptides (lysine/arginine amino acids) over total peptides. The number of detected peptides is shown on the y axis and the percentage of heavy labeled peptides over total peptides (heavy and light) on the x axis. MN cultures with mutSOD1 are shown in red and isogenic controls in green. (C) Quantification of the total number of labeled and unlabeled peptides in both mutSOD1 and isogenic control MN cultures shown over time in culture. (D) Normalized mean peptide intensity of all labeled proteins detected in both mutSOD1 and isogenic control MNs within each one of the three time points interrogated. All values are normalized to the respective values on day 30. Each dot represents a single protein, and average and standard deviation values are shown for each time point. On day 31 (24 h after ‘‘chase’’) there were 39 common proteins with a mean intensity of 0.62 and 0.42 in mutSOD1 and isogenic control MNs, respectively. On day 35 (5 days after ‘‘chase’’) there were 87 common proteins with a mean intensity of 0.44 and 0.38 in mutSOD1 and isogenic control MNs, respectively. On day 51 (21 days after ‘‘chase’’) there were 129 common proteins with a mean intensity of 0.20 and 0.15 in mutSOD1 and isogenic control MNs, respectively. Paired t test (two-tailed), day 31 ****p < 0.0001, day 35 **p = 0.0053, day 51 **p = 0.0012. (E) Venn diagram of the number of proteins that are more labeled (i.e., persist) in SOD1+/A4V MN cultures relative to isogenic controls across all three time points (days 31, 35, and 51). The eight proteins that are more labeled across all the three time points examined are highlighted.

Journal: Cell reports

Article Title: Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.

doi: 10.1016/j.celrep.2023.113160

Figure Lengend Snippet: Figure 2. Mutant SOD1 iPSC-derived patient neurons exhibit slower protein degradation dynamics of a small panel of proteins including VCP/p97 (A) Experimental schematic of SILAC-MS pulse-chase strategy for iPSC-MNs. (B) Assessment of labeling efficiency on day-30 MNs based on the quantification of heavy labeled peptides (lysine/arginine amino acids) over total peptides. The number of detected peptides is shown on the y axis and the percentage of heavy labeled peptides over total peptides (heavy and light) on the x axis. MN cultures with mutSOD1 are shown in red and isogenic controls in green. (C) Quantification of the total number of labeled and unlabeled peptides in both mutSOD1 and isogenic control MN cultures shown over time in culture. (D) Normalized mean peptide intensity of all labeled proteins detected in both mutSOD1 and isogenic control MNs within each one of the three time points interrogated. All values are normalized to the respective values on day 30. Each dot represents a single protein, and average and standard deviation values are shown for each time point. On day 31 (24 h after ‘‘chase’’) there were 39 common proteins with a mean intensity of 0.62 and 0.42 in mutSOD1 and isogenic control MNs, respectively. On day 35 (5 days after ‘‘chase’’) there were 87 common proteins with a mean intensity of 0.44 and 0.38 in mutSOD1 and isogenic control MNs, respectively. On day 51 (21 days after ‘‘chase’’) there were 129 common proteins with a mean intensity of 0.20 and 0.15 in mutSOD1 and isogenic control MNs, respectively. Paired t test (two-tailed), day 31 ****p < 0.0001, day 35 **p = 0.0053, day 51 **p = 0.0012. (E) Venn diagram of the number of proteins that are more labeled (i.e., persist) in SOD1+/A4V MN cultures relative to isogenic controls across all three time points (days 31, 35, and 51). The eight proteins that are more labeled across all the three time points examined are highlighted.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-VCP mouse primary antibody Genetex GTX101089; RRID:AB_1952544 Anti-SOD1 rabbit primary antibody Abcam ab51254; RRID:AB_882757 Anti-MAP2 chicken primary antibody Abcam ab5392; RRID:AB_2138153 Anti-b-Tubulin III rabbit primary antibody Millipore-Sigma T2200-200UL; RRID:AB_262133 Anti-Choline Acetyltransferase goat primary antibody Millipore-Sigma AB144P-200UL; RRID:AB_2079751 Anti-ISL1/2 mouse primary antibody DSHB (University of Iowa) 39.4D5; RRID:AB_2314683 ms SOD1 134.2 (57–72) Umeå University Zetterstrom et al., 2011 rb SOD1 (24–39) Umeå University Zetterstrom et al., 2011 VCP Abcam ab109240; RRID:AB_10862588 Ub (P4D1) Santa-Cruz Biotechnology sc-8017; RRID:AB_628423 p62/SQSTM1 Proteintech 18420-1-AP; RRID:AB_10694431 HSPB1 Proteintech 18284-1-AP; RRID:AB_2295540 Normal Rabbit IgG Cell Signaling 2729S; RRID:AB_1031062 Myc-Tag (9B11) Cell Signaling 2276S; RRID:AB_331783 Alexa Fluor 488 goat anti-mouse secondary antibody Jackson Immuno Research (Fisher Scientific) A11001; RRID:AB_2534069 Alexa Fluor 594 donkey anti-rabbit secondary antibody Jackson Immuno Research (Fisher Scientific) A21207; RRID:AB_141637 Alexa Fluor 647 donkey anti-chicken secondary antibody Jackson Immuno Research (Fisher Scientific) 703-606-155; RRID:AB_2340380 Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 Invitrogen A21202; RRID:AB_141607 Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 Invitrogen A11055; RRID:AB_2534102 Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 647 Invitrogen A31573; RRID:AB_2536183 DAPI Invitrogen H21492 Cell Culture Reagents mTeSR Stem Cell Technologies 5820 N-2 Supplement (100X) Thermo Fisher Scientific 17502001 B-27 Supplement (50X), serum free Thermo Fisher Scientific 17504001 GlutaMAX Supplement Thermo Fisher Scientific 35050061 MEM Non-Essential Amino Acids Solution (100X) Thermo Fisher Scientific 11140050 DMEM/F-12 Thermo Fisher Scientific 11320082 Neurobasal Medium Thermo Fisher Scientific 21103049 EdU Thermo Fisher Scientific A10044 Trypsin-EDTA (0.25%), phenol red Thermo Fisher Scientific 25200072 TrypLETM Express Enzyme (1X), no phenol red Thermo Fisher Scientific 12604013 1X PBS Corning MT21040CV Ultrapure water with 0.1% Gelatin Millipore ES006B Accutase Innovative Cell Technologies AT 104-500 SB431542 DNSK International DNSK-KI-12 SAG DNSK International DNSK-SMO-1 SU5402 DNSK International DNSK-KI-11 (Continued on next page) 18 Cell Reports 42, 113160, October 31, 2023

Techniques: Mutagenesis, Derivative Assay, Multiplex sample analysis, Pulse Chase, Labeling, Control, Standard Deviation, Two Tailed Test

Figure 3. The SOD1 A4V mutation is sufficient to alter VCP turnover (A) Experimental schematic of the HUES3 stem cell editing and MN-directed differentiation. (B) Representative images of stem-cell-differentiated MNs expressing ISL1/2 and TUJ1 on day 25. Scale bar, 10 mm. (C and D) WB analysis (C) and quantification (D) of the detergent-insoluble SOD1 levels in HUES3-SOD1+/+ or HUES3-SOD1+/A4V MNs across time. The detergent-insoluble SOD1 (top blot) is expressed relative to the normalized soluble SOD1 (bottom blot). Two-way ANOVA across time and genotypes, ****p < 0.0001; Sidak’s multiple comparisons test per time point, day 16 p = 0.9571, day 25 ***p = 0.0008, day 30 **p = 0.0033, day 35 **p = 0.0037, day 40 ****p < 0.0001, day 51 ****p < 0.0001; ns, not significant; n = 3 independent differentiations. (E) Schematic representation of the SILAC-IP LC-MS/MS approach. (F) Average intensity of heavy VCP peptides that are enriched in both HUES3-SOD1+/+ and HUES3-SOD1+/A4V MN cultures on day 51 upon immunoprecipitation. The comparison is done between identical, common VCP peptides in both genotypes. Paired t test, Wilcoxon correction; n = 2 independent differentiations; experiment 1, ****p < 0.0001; experiment 2, **p = 0.009.

Journal: Cell reports

Article Title: Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.

doi: 10.1016/j.celrep.2023.113160

Figure Lengend Snippet: Figure 3. The SOD1 A4V mutation is sufficient to alter VCP turnover (A) Experimental schematic of the HUES3 stem cell editing and MN-directed differentiation. (B) Representative images of stem-cell-differentiated MNs expressing ISL1/2 and TUJ1 on day 25. Scale bar, 10 mm. (C and D) WB analysis (C) and quantification (D) of the detergent-insoluble SOD1 levels in HUES3-SOD1+/+ or HUES3-SOD1+/A4V MNs across time. The detergent-insoluble SOD1 (top blot) is expressed relative to the normalized soluble SOD1 (bottom blot). Two-way ANOVA across time and genotypes, ****p < 0.0001; Sidak’s multiple comparisons test per time point, day 16 p = 0.9571, day 25 ***p = 0.0008, day 30 **p = 0.0033, day 35 **p = 0.0037, day 40 ****p < 0.0001, day 51 ****p < 0.0001; ns, not significant; n = 3 independent differentiations. (E) Schematic representation of the SILAC-IP LC-MS/MS approach. (F) Average intensity of heavy VCP peptides that are enriched in both HUES3-SOD1+/+ and HUES3-SOD1+/A4V MN cultures on day 51 upon immunoprecipitation. The comparison is done between identical, common VCP peptides in both genotypes. Paired t test, Wilcoxon correction; n = 2 independent differentiations; experiment 1, ****p < 0.0001; experiment 2, **p = 0.009.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-VCP mouse primary antibody Genetex GTX101089; RRID:AB_1952544 Anti-SOD1 rabbit primary antibody Abcam ab51254; RRID:AB_882757 Anti-MAP2 chicken primary antibody Abcam ab5392; RRID:AB_2138153 Anti-b-Tubulin III rabbit primary antibody Millipore-Sigma T2200-200UL; RRID:AB_262133 Anti-Choline Acetyltransferase goat primary antibody Millipore-Sigma AB144P-200UL; RRID:AB_2079751 Anti-ISL1/2 mouse primary antibody DSHB (University of Iowa) 39.4D5; RRID:AB_2314683 ms SOD1 134.2 (57–72) Umeå University Zetterstrom et al., 2011 rb SOD1 (24–39) Umeå University Zetterstrom et al., 2011 VCP Abcam ab109240; RRID:AB_10862588 Ub (P4D1) Santa-Cruz Biotechnology sc-8017; RRID:AB_628423 p62/SQSTM1 Proteintech 18420-1-AP; RRID:AB_10694431 HSPB1 Proteintech 18284-1-AP; RRID:AB_2295540 Normal Rabbit IgG Cell Signaling 2729S; RRID:AB_1031062 Myc-Tag (9B11) Cell Signaling 2276S; RRID:AB_331783 Alexa Fluor 488 goat anti-mouse secondary antibody Jackson Immuno Research (Fisher Scientific) A11001; RRID:AB_2534069 Alexa Fluor 594 donkey anti-rabbit secondary antibody Jackson Immuno Research (Fisher Scientific) A21207; RRID:AB_141637 Alexa Fluor 647 donkey anti-chicken secondary antibody Jackson Immuno Research (Fisher Scientific) 703-606-155; RRID:AB_2340380 Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 Invitrogen A21202; RRID:AB_141607 Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 Invitrogen A11055; RRID:AB_2534102 Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 647 Invitrogen A31573; RRID:AB_2536183 DAPI Invitrogen H21492 Cell Culture Reagents mTeSR Stem Cell Technologies 5820 N-2 Supplement (100X) Thermo Fisher Scientific 17502001 B-27 Supplement (50X), serum free Thermo Fisher Scientific 17504001 GlutaMAX Supplement Thermo Fisher Scientific 35050061 MEM Non-Essential Amino Acids Solution (100X) Thermo Fisher Scientific 11140050 DMEM/F-12 Thermo Fisher Scientific 11320082 Neurobasal Medium Thermo Fisher Scientific 21103049 EdU Thermo Fisher Scientific A10044 Trypsin-EDTA (0.25%), phenol red Thermo Fisher Scientific 25200072 TrypLETM Express Enzyme (1X), no phenol red Thermo Fisher Scientific 12604013 1X PBS Corning MT21040CV Ultrapure water with 0.1% Gelatin Millipore ES006B Accutase Innovative Cell Technologies AT 104-500 SB431542 DNSK International DNSK-KI-12 SAG DNSK International DNSK-SMO-1 SU5402 DNSK International DNSK-KI-11 (Continued on next page) 18 Cell Reports 42, 113160, October 31, 2023

Techniques: Mutagenesis, Expressing, Multiplex sample analysis, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Comparison

Figure 4. Accumulated VCP in postmortem tissue of an SOD1+/A4V

Journal: Cell reports

Article Title: Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.

doi: 10.1016/j.celrep.2023.113160

Figure Lengend Snippet: Figure 4. Accumulated VCP in postmortem tissue of an SOD1+/A4V

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-VCP mouse primary antibody Genetex GTX101089; RRID:AB_1952544 Anti-SOD1 rabbit primary antibody Abcam ab51254; RRID:AB_882757 Anti-MAP2 chicken primary antibody Abcam ab5392; RRID:AB_2138153 Anti-b-Tubulin III rabbit primary antibody Millipore-Sigma T2200-200UL; RRID:AB_262133 Anti-Choline Acetyltransferase goat primary antibody Millipore-Sigma AB144P-200UL; RRID:AB_2079751 Anti-ISL1/2 mouse primary antibody DSHB (University of Iowa) 39.4D5; RRID:AB_2314683 ms SOD1 134.2 (57–72) Umeå University Zetterstrom et al., 2011 rb SOD1 (24–39) Umeå University Zetterstrom et al., 2011 VCP Abcam ab109240; RRID:AB_10862588 Ub (P4D1) Santa-Cruz Biotechnology sc-8017; RRID:AB_628423 p62/SQSTM1 Proteintech 18420-1-AP; RRID:AB_10694431 HSPB1 Proteintech 18284-1-AP; RRID:AB_2295540 Normal Rabbit IgG Cell Signaling 2729S; RRID:AB_1031062 Myc-Tag (9B11) Cell Signaling 2276S; RRID:AB_331783 Alexa Fluor 488 goat anti-mouse secondary antibody Jackson Immuno Research (Fisher Scientific) A11001; RRID:AB_2534069 Alexa Fluor 594 donkey anti-rabbit secondary antibody Jackson Immuno Research (Fisher Scientific) A21207; RRID:AB_141637 Alexa Fluor 647 donkey anti-chicken secondary antibody Jackson Immuno Research (Fisher Scientific) 703-606-155; RRID:AB_2340380 Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 Invitrogen A21202; RRID:AB_141607 Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 Invitrogen A11055; RRID:AB_2534102 Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 647 Invitrogen A31573; RRID:AB_2536183 DAPI Invitrogen H21492 Cell Culture Reagents mTeSR Stem Cell Technologies 5820 N-2 Supplement (100X) Thermo Fisher Scientific 17502001 B-27 Supplement (50X), serum free Thermo Fisher Scientific 17504001 GlutaMAX Supplement Thermo Fisher Scientific 35050061 MEM Non-Essential Amino Acids Solution (100X) Thermo Fisher Scientific 11140050 DMEM/F-12 Thermo Fisher Scientific 11320082 Neurobasal Medium Thermo Fisher Scientific 21103049 EdU Thermo Fisher Scientific A10044 Trypsin-EDTA (0.25%), phenol red Thermo Fisher Scientific 25200072 TrypLETM Express Enzyme (1X), no phenol red Thermo Fisher Scientific 12604013 1X PBS Corning MT21040CV Ultrapure water with 0.1% Gelatin Millipore ES006B Accutase Innovative Cell Technologies AT 104-500 SB431542 DNSK International DNSK-KI-12 SAG DNSK International DNSK-SMO-1 SU5402 DNSK International DNSK-KI-11 (Continued on next page) 18 Cell Reports 42, 113160, October 31, 2023

Techniques:

Figure 6. VCP impacts the solubility of SOD1 (A) Experimental workflow of HEK293T transfection and treatment. HEK293T cells were transfected with SOD1WT-MYC or SOD1A4V-MYC plasmids in com- bination with VCP-RFP or RFP plasmids. The cells were incubated up to 48 h and treated with the allosteric VCP inhibitor NMS873 (10 mM, 8 h). The cells were lysed and subjected to fractionation for biochemical analysis. (B) WB analysis of HEK293T-transfected cells. Lanes 1–5 correspond to DMSO-treated cells and lanes 6–10 to NMS873-treated cells. The bar graph corresponds to the levels of the detergent-insoluble SOD1-MYC (insoluble fraction, bottom blot). The detergent-insoluble SOD1-MYC is normalized to the soluble SOD1-MYC levels (soluble fraction, top blot); n = 2 independent transfections. (C) Schematic representation of patient or isogenic control MNs treated with NMS873 (10 mM, 8 h) on day 35 and lysed for biochemical analysis. (D and E) WB analysis and quantification of whole-cell extracts from DMSO- or NMS873-treated MNs and quantification of poly-Ub (D) or SOD1 (E) protein levels. For poly-Ub, two-way ANOVA (treatment 3 genotype), *p = 0.0152; Sidak’s multiple comparisons test per treatment, SOD1+/A4V MN ****p < 0.0001, SOD1+/+ MN p = 0.396. For SOD1 two-way ANOVA (treatment 3 genotype), p = 0.2170; Sidak’s multiple comparisons test per treatment, SOD1+/A4V MN **p = 0.0099, SOD1+/+

Journal: Cell reports

Article Title: Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.

doi: 10.1016/j.celrep.2023.113160

Figure Lengend Snippet: Figure 6. VCP impacts the solubility of SOD1 (A) Experimental workflow of HEK293T transfection and treatment. HEK293T cells were transfected with SOD1WT-MYC or SOD1A4V-MYC plasmids in com- bination with VCP-RFP or RFP plasmids. The cells were incubated up to 48 h and treated with the allosteric VCP inhibitor NMS873 (10 mM, 8 h). The cells were lysed and subjected to fractionation for biochemical analysis. (B) WB analysis of HEK293T-transfected cells. Lanes 1–5 correspond to DMSO-treated cells and lanes 6–10 to NMS873-treated cells. The bar graph corresponds to the levels of the detergent-insoluble SOD1-MYC (insoluble fraction, bottom blot). The detergent-insoluble SOD1-MYC is normalized to the soluble SOD1-MYC levels (soluble fraction, top blot); n = 2 independent transfections. (C) Schematic representation of patient or isogenic control MNs treated with NMS873 (10 mM, 8 h) on day 35 and lysed for biochemical analysis. (D and E) WB analysis and quantification of whole-cell extracts from DMSO- or NMS873-treated MNs and quantification of poly-Ub (D) or SOD1 (E) protein levels. For poly-Ub, two-way ANOVA (treatment 3 genotype), *p = 0.0152; Sidak’s multiple comparisons test per treatment, SOD1+/A4V MN ****p < 0.0001, SOD1+/+ MN p = 0.396. For SOD1 two-way ANOVA (treatment 3 genotype), p = 0.2170; Sidak’s multiple comparisons test per treatment, SOD1+/A4V MN **p = 0.0099, SOD1+/+

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-VCP mouse primary antibody Genetex GTX101089; RRID:AB_1952544 Anti-SOD1 rabbit primary antibody Abcam ab51254; RRID:AB_882757 Anti-MAP2 chicken primary antibody Abcam ab5392; RRID:AB_2138153 Anti-b-Tubulin III rabbit primary antibody Millipore-Sigma T2200-200UL; RRID:AB_262133 Anti-Choline Acetyltransferase goat primary antibody Millipore-Sigma AB144P-200UL; RRID:AB_2079751 Anti-ISL1/2 mouse primary antibody DSHB (University of Iowa) 39.4D5; RRID:AB_2314683 ms SOD1 134.2 (57–72) Umeå University Zetterstrom et al., 2011 rb SOD1 (24–39) Umeå University Zetterstrom et al., 2011 VCP Abcam ab109240; RRID:AB_10862588 Ub (P4D1) Santa-Cruz Biotechnology sc-8017; RRID:AB_628423 p62/SQSTM1 Proteintech 18420-1-AP; RRID:AB_10694431 HSPB1 Proteintech 18284-1-AP; RRID:AB_2295540 Normal Rabbit IgG Cell Signaling 2729S; RRID:AB_1031062 Myc-Tag (9B11) Cell Signaling 2276S; RRID:AB_331783 Alexa Fluor 488 goat anti-mouse secondary antibody Jackson Immuno Research (Fisher Scientific) A11001; RRID:AB_2534069 Alexa Fluor 594 donkey anti-rabbit secondary antibody Jackson Immuno Research (Fisher Scientific) A21207; RRID:AB_141637 Alexa Fluor 647 donkey anti-chicken secondary antibody Jackson Immuno Research (Fisher Scientific) 703-606-155; RRID:AB_2340380 Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 Invitrogen A21202; RRID:AB_141607 Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 Invitrogen A11055; RRID:AB_2534102 Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 647 Invitrogen A31573; RRID:AB_2536183 DAPI Invitrogen H21492 Cell Culture Reagents mTeSR Stem Cell Technologies 5820 N-2 Supplement (100X) Thermo Fisher Scientific 17502001 B-27 Supplement (50X), serum free Thermo Fisher Scientific 17504001 GlutaMAX Supplement Thermo Fisher Scientific 35050061 MEM Non-Essential Amino Acids Solution (100X) Thermo Fisher Scientific 11140050 DMEM/F-12 Thermo Fisher Scientific 11320082 Neurobasal Medium Thermo Fisher Scientific 21103049 EdU Thermo Fisher Scientific A10044 Trypsin-EDTA (0.25%), phenol red Thermo Fisher Scientific 25200072 TrypLETM Express Enzyme (1X), no phenol red Thermo Fisher Scientific 12604013 1X PBS Corning MT21040CV Ultrapure water with 0.1% Gelatin Millipore ES006B Accutase Innovative Cell Technologies AT 104-500 SB431542 DNSK International DNSK-KI-12 SAG DNSK International DNSK-SMO-1 SU5402 DNSK International DNSK-KI-11 (Continued on next page) 18 Cell Reports 42, 113160, October 31, 2023

Techniques: Solubility, Transfection, Incubation, Fractionation, Control

Figure 7. VCP ameliorates mutSOD1 toxicity in iPSC-MNs in vitro and C. elegans models in vivo (A) Experimental schematic of MN viability assay. MNs were differentiated from mutSOD1 and isogenic control iPSCs and infected with either LV-VCP-T2A-RFP or LV-RFP to monitor progressive degeneration by longitudinal time-lapse imaging microscopy. (B) Probability of survival of mutSOD1 and isogenic control iMNs upon VCP-RFP or RFP expression across 3 weeks in culture; n = 1 differentiation and 3 technical replicates; for SOD1+/A4V iMN LV-VCP-T2A-RFP n = 220, LV-RFP n = 197; for SOD1+/+ iMN LV-VCP-T2A-RFP n = 200, LV-RFP n = 198; Gehan- Breslow-Wilcoxon test. (C) Experimental schematic of genetic interaction experiments with C. elegans strains expressing human mutSOD1 protein (IW8), with overexpression (o-e) and knockout (ko) of the VCP ortholog cdc48.1. (D) Quantification of the average speed per second of C. elegans strains examined. One-way ANOVA for genotype, p < 0.0001; Unpaired t test to compare individual genotypes: (1) WT vs. mutSOD1 *p = 0.0206, (2) WT vs. ko VCP **p = 0.0042, (3) WT vs. o-e VCP p = 0.2096, (4) WT vs. mutSOD1; ko VCP **p = 0.0013, (5) WT vs. mutSOD1; o-e VCP p = 0.7650, (6) mutSOD1; o-e VCP vs. mutSOD1; ko VCP **p = 0.0023, (7) mutSOD1; o-e VCP vs. o-e VCP p = 0.1325, (8) mutSOD1; o-e VCP vs. ko VCP **p = 0.0044, (9) mutSOD1; o-e VCP vs. mutSOD1 *p = 0.0154; n = 3–5 independent experiments, n = 5–10 worms per experiment. (E) Quantification of SOD1 protein within the insoluble and soluble fractions in mutSOD1 worms and mutSOD1 overexpressing VCP. The amount of detergent- insoluble SOD1 is expressed relative to the amount of normalized soluble SOD1. Unpaired t test (two-tailed), *p = 0.0123; n = 4 independent preparations of worm cultures.

Journal: Cell reports

Article Title: Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.

doi: 10.1016/j.celrep.2023.113160

Figure Lengend Snippet: Figure 7. VCP ameliorates mutSOD1 toxicity in iPSC-MNs in vitro and C. elegans models in vivo (A) Experimental schematic of MN viability assay. MNs were differentiated from mutSOD1 and isogenic control iPSCs and infected with either LV-VCP-T2A-RFP or LV-RFP to monitor progressive degeneration by longitudinal time-lapse imaging microscopy. (B) Probability of survival of mutSOD1 and isogenic control iMNs upon VCP-RFP or RFP expression across 3 weeks in culture; n = 1 differentiation and 3 technical replicates; for SOD1+/A4V iMN LV-VCP-T2A-RFP n = 220, LV-RFP n = 197; for SOD1+/+ iMN LV-VCP-T2A-RFP n = 200, LV-RFP n = 198; Gehan- Breslow-Wilcoxon test. (C) Experimental schematic of genetic interaction experiments with C. elegans strains expressing human mutSOD1 protein (IW8), with overexpression (o-e) and knockout (ko) of the VCP ortholog cdc48.1. (D) Quantification of the average speed per second of C. elegans strains examined. One-way ANOVA for genotype, p < 0.0001; Unpaired t test to compare individual genotypes: (1) WT vs. mutSOD1 *p = 0.0206, (2) WT vs. ko VCP **p = 0.0042, (3) WT vs. o-e VCP p = 0.2096, (4) WT vs. mutSOD1; ko VCP **p = 0.0013, (5) WT vs. mutSOD1; o-e VCP p = 0.7650, (6) mutSOD1; o-e VCP vs. mutSOD1; ko VCP **p = 0.0023, (7) mutSOD1; o-e VCP vs. o-e VCP p = 0.1325, (8) mutSOD1; o-e VCP vs. ko VCP **p = 0.0044, (9) mutSOD1; o-e VCP vs. mutSOD1 *p = 0.0154; n = 3–5 independent experiments, n = 5–10 worms per experiment. (E) Quantification of SOD1 protein within the insoluble and soluble fractions in mutSOD1 worms and mutSOD1 overexpressing VCP. The amount of detergent- insoluble SOD1 is expressed relative to the amount of normalized soluble SOD1. Unpaired t test (two-tailed), *p = 0.0123; n = 4 independent preparations of worm cultures.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-VCP mouse primary antibody Genetex GTX101089; RRID:AB_1952544 Anti-SOD1 rabbit primary antibody Abcam ab51254; RRID:AB_882757 Anti-MAP2 chicken primary antibody Abcam ab5392; RRID:AB_2138153 Anti-b-Tubulin III rabbit primary antibody Millipore-Sigma T2200-200UL; RRID:AB_262133 Anti-Choline Acetyltransferase goat primary antibody Millipore-Sigma AB144P-200UL; RRID:AB_2079751 Anti-ISL1/2 mouse primary antibody DSHB (University of Iowa) 39.4D5; RRID:AB_2314683 ms SOD1 134.2 (57–72) Umeå University Zetterstrom et al., 2011 rb SOD1 (24–39) Umeå University Zetterstrom et al., 2011 VCP Abcam ab109240; RRID:AB_10862588 Ub (P4D1) Santa-Cruz Biotechnology sc-8017; RRID:AB_628423 p62/SQSTM1 Proteintech 18420-1-AP; RRID:AB_10694431 HSPB1 Proteintech 18284-1-AP; RRID:AB_2295540 Normal Rabbit IgG Cell Signaling 2729S; RRID:AB_1031062 Myc-Tag (9B11) Cell Signaling 2276S; RRID:AB_331783 Alexa Fluor 488 goat anti-mouse secondary antibody Jackson Immuno Research (Fisher Scientific) A11001; RRID:AB_2534069 Alexa Fluor 594 donkey anti-rabbit secondary antibody Jackson Immuno Research (Fisher Scientific) A21207; RRID:AB_141637 Alexa Fluor 647 donkey anti-chicken secondary antibody Jackson Immuno Research (Fisher Scientific) 703-606-155; RRID:AB_2340380 Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 Invitrogen A21202; RRID:AB_141607 Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa FluorTM 488 Invitrogen A11055; RRID:AB_2534102 Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM 647 Invitrogen A31573; RRID:AB_2536183 DAPI Invitrogen H21492 Cell Culture Reagents mTeSR Stem Cell Technologies 5820 N-2 Supplement (100X) Thermo Fisher Scientific 17502001 B-27 Supplement (50X), serum free Thermo Fisher Scientific 17504001 GlutaMAX Supplement Thermo Fisher Scientific 35050061 MEM Non-Essential Amino Acids Solution (100X) Thermo Fisher Scientific 11140050 DMEM/F-12 Thermo Fisher Scientific 11320082 Neurobasal Medium Thermo Fisher Scientific 21103049 EdU Thermo Fisher Scientific A10044 Trypsin-EDTA (0.25%), phenol red Thermo Fisher Scientific 25200072 TrypLETM Express Enzyme (1X), no phenol red Thermo Fisher Scientific 12604013 1X PBS Corning MT21040CV Ultrapure water with 0.1% Gelatin Millipore ES006B Accutase Innovative Cell Technologies AT 104-500 SB431542 DNSK International DNSK-KI-12 SAG DNSK International DNSK-SMO-1 SU5402 DNSK International DNSK-KI-11 (Continued on next page) 18 Cell Reports 42, 113160, October 31, 2023

Techniques: In Vitro, In Vivo, Viability Assay, Control, Infection, Imaging, Microscopy, Expressing, Over Expression, Knock-Out, Two Tailed Test