Journal: The Journal of Cell Biology

Article Title: Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

doi: 10.1083/jcb.202312141

Figure Lengend Snippet: CTLA4 expression in cancer cell lines. (A) mRNA levels derived from the Cancer Cell Line Encyclopedia (CCLE) database . Each dot represents an individual tumor cell line within the indicated categories. (B) Representative western blot of NCI-H520 and A2058 cells transfected with siRNA against non-targeting (NT1) or CTLA4 for 72 h prior to lysis. (C) Representative western blot of HeLa S3 Flp-In CTLA4-HA cells treated with Dynasore (80 µM) for 30 min in serum-free media before the addition of Cycloheximide (CHX, 100 µg/ml) for 2 h. (D) Quantification of CTLA4-HA levels for data represented in C. Individual data points from three independent, color-coded experiments are shown. Error bars indicate SD. One-way ANOVA and Dunnett’s multiple comparison test, **P < 0.01, ****P < 0.0001. Source data are available for this figure: .

Article Snippet: Human CTLA4-HA DNA flanked by BglII/EcoRI sites was synthesized and provided in a pTWIST-High-Kan vector by TWIST Bioscience, and subcloned into pcDNA3.1 and pEF5/FRT plasmids.

Techniques: Expressing, Derivative Assay, Western Blot, Transfection, Lysis, Comparison

Journal: The Journal of Cell Biology

Article Title: Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

doi: 10.1083/jcb.202312141

Figure Lengend Snippet: Lysosomal degradation of CTLA4 requires a functional ubiquitin conjugation cascade. (A) Representative western blots of A2058, NCI-H520, and HeLa S3 Flp-In CTLA4-HA cells treated with Cycloheximide (CHX, 100 µg/ml) for indicated times prior to lysis. (B) Quantification of data represented in A. The half-life of CTLA4 in different cell lines was estimated using an exponential decay model. Error bars indicate SD from three independent experiments. (C and E) Representative western blots of A2058 and NCI-H520 cells treated with Concanamycin (ConcA, 100 nM), Epoxomicin (Epo, 100 nM), or TAK-243 (TAK, 1 µM) for 15 min before the addition of CHX for 1 h (C, A2058) or 2 h (E, NCI-H520). (D and F) Quantification of CTLA4 levels after treatment with indicated inhibitors relative to control for data represented in C and E. Individual data points from three independent, color-coded experiments are shown. Error bars show SD. One-way ANOVA and Dunnett’s multiple comparisons test, *P < 0.05, **P < 0.01, ****P < 0.0001. Source data are available for this figure: .

Article Snippet: Human CTLA4-HA DNA flanked by BglII/EcoRI sites was synthesized and provided in a pTWIST-High-Kan vector by TWIST Bioscience, and subcloned into pcDNA3.1 and pEF5/FRT plasmids.

Techniques: Functional Assay, Ubiquitin Proteomics, Conjugation Assay, Western Blot, Lysis, Control

Journal: The Journal of Cell Biology

Article Title: Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

doi: 10.1083/jcb.202312141

Figure Lengend Snippet: CTLA4 is secreted via exosomes from A2058 and HeLa S3 Flp-In CTLA4-HA cells. (A and B) Representative western blots of the lysates and cultured supernatants from mock- and Concanamycin A (ConcA)-treated A2058 and HeLa S3 Flp-In CTLA4-HA cells. Cells were lysed and media were subjected to trichloroacetic acid (TCA) precipitation. (C) Representative western blots of the lysates and cultured supernatants from mock- and ConcA-treated HeLa S3 Flp-In CTLA4-HA cells for indicated times. Cells were lysed and the media was collected by serial centrifugation to concentrate extracellular vesicles (100k pellet, exosome fraction). (D) Quantification of CTLA4, ALIX, Syntenin and CD63 secreted in the exosome fractions (100k) for data represented in C. Individual data points from three independent, color-coded experiments are shown. Error bars show SD. (E) Representative western blot of Cycloheximide (CHX; 100 µg/ml) chase experiment in HeLa S3 Flp-In CTLA4-HA and A2058 cells, following treatment with non-targeting (NT1) or ATG7 siRNA for 72 h. * indicates a non-specific band. (F) Quantification of blots represented in E. Error bars show SD from three independent experiments. Source data are available for this figure: .

Article Snippet: Human CTLA4-HA DNA flanked by BglII/EcoRI sites was synthesized and provided in a pTWIST-High-Kan vector by TWIST Bioscience, and subcloned into pcDNA3.1 and pEF5/FRT plasmids.

Techniques: Western Blot, Cell Culture, TCA Precipitation, Centrifugation

Journal: The Journal of Cell Biology

Article Title: Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

doi: 10.1083/jcb.202312141

Figure Lengend Snippet: CTLA4 degradation is delayed in the absence of USP8. (A–F) Representative western blots and associated quantifications of Cycloheximide (CHX; 100 µg/ml) chase experiments in HeLa S3 Flp-In CTLA4-HA (A, C, and E) and A2058 (B, D, and F) cells following treatment with non-targeting (NT1), AMSH, USP8, or HRS siRNA for 72 h. The half-life of CTLA4 was estimated using an exponential decay model. Error bars show SD from three independent experiments. Source data are available for this figure: .

Article Snippet: Human CTLA4-HA DNA flanked by BglII/EcoRI sites was synthesized and provided in a pTWIST-High-Kan vector by TWIST Bioscience, and subcloned into pcDNA3.1 and pEF5/FRT plasmids.

Techniques: Western Blot

Journal: The Journal of Cell Biology

Article Title: Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

doi: 10.1083/jcb.202312141

Figure Lengend Snippet: USP8 depletion dramatically enhances CTLA4 ubiquitylation. (A) Representative western blots of TUBES pulldown of ubiquitylated CTLA4. HeLa S3 Flp-In CTLA4-HA and A2058 cells were transfected for 72 h with non-targeting (NT1), USP8, and AMSH siRNA prior to lysis. Lysates were subjected to TUBES-pulldown (IB: Immunoblot). (B) Quantification of ubiquitylated CTLA4-HA isolated by TUBES pulldown for data represented in A. Ubiquitylated CTLA4-HA was normalized to total ubiquitin pulled down. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (C) Representative western blots of ubiquitylated CTLA4-HA immunoprecipitated under denaturing conditions. HeLa S3 Flp-In parental (Par) or CTLA4-HA (HA) cells were transfected for 72 h with non-targeting (NT1), USP8, and AMSH siRNA. Cells were lysed in denaturing SDS lysis buffer and lysates were subjected to immunoprecipitation (IP) with anti-HA coupled magnetic beads. *Antibody heavy chain; ** antibody light chain. (D) Quantification of CTLA4-HA ubiquitylation relative to NT1 for data represented in C. Ubiquitylated CTLA4-HA was normalized to total immunoprecipitated CTLA4-HA. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (E) Representative western blots of TUBES pulldown of ubiquitylated CTLA4. Lysates from USP8 fl/fl (Control, n = 3) and USP8 deleted (∆USP8, n = 3) activated T cells derived from individual mice were either analyzed directly by SDS-PAGE and western blot (left blot), or first subjected to a TUBES pulldown prior to analysis alongside input samples. (F) Quantification of ubiquitylated CTLA4 enriched by TUBES pulldown for data represented in E. Ubiquitylated CTLA4 was ratioed to total CTLA4 levels. Individual data points from six individual mice are shown. Error bars indicate SD. Source data are available for this figure: .

Article Snippet: Human CTLA4-HA DNA flanked by BglII/EcoRI sites was synthesized and provided in a pTWIST-High-Kan vector by TWIST Bioscience, and subcloned into pcDNA3.1 and pEF5/FRT plasmids.

Techniques: Western Blot, Transfection, Lysis, Isolation, Ubiquitin Proteomics, Immunoprecipitation, Magnetic Beads, Control, Derivative Assay, SDS Page

Journal: The Journal of Cell Biology

Article Title: Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

doi: 10.1083/jcb.202312141

Figure Lengend Snippet: AP2 depletion delays CTLA4 turnover and reduces CTLA4 ubiquitylation in the absence of USP8. (A and D) Representative western blots following treatment with Cycloheximide (CHX; 100 µg/ml) for indicated times in HeLa S3 Flp-In CTLA4-HA (A) and A2058 (D) transfected with indicated siRNAs. Cells were transfected with non-targeting (NT1) or AP2 siRNA targeting the µ2 subunit and reseeded for a second round of transfection 24 h later with NT1, AP2, and/or USP8 siRNA. The cells were left for a further 72 h before lysis. The last lane represents untransfected control. *non-specific signal. (B and E) Quantification of basal CTLA4 levels normalized to untransfected control for data represented in A and D. Error bars show SD from three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test, *P < 0.05, **P < 0.01. (C and F) Quantification of CTLA4 levels following treatment with Cycloheximide for data represented in A and D. One-way ANOVA with Dunnett’s multiple comparisons test, *P < 0.05, **P < 0.01. (G and H) Representative western blots of TUBES pulldown of ubiquitylated CTLA4. HeLa S3 Flp-In CTLA4-HA and A2058 cells were transfected as in A and D. IB: immunoblot. *non-specific signal. (I and J) Quantification of ubiquitylated CTLA4 relative to total CTLA4 levels for data represented in G and H. Individual data points from two (I) or three (J) independent, color-coded experiments are shown. Error bars indicate range ( n = 2) or SD ( n = 3). Source data are available for this figure: .

Article Snippet: Human CTLA4-HA DNA flanked by BglII/EcoRI sites was synthesized and provided in a pTWIST-High-Kan vector by TWIST Bioscience, and subcloned into pcDNA3.1 and pEF5/FRT plasmids.

Techniques: Western Blot, Transfection, Lysis, Control

Journal: The Journal of Cell Biology

Article Title: Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

doi: 10.1083/jcb.202312141

Figure Lengend Snippet: CTLA4 accumulates in enlarged endosomes following USP8 depletion. (A and B) Representative confocal images of CTLA4-HA co-stained for EEA1 (A) or LAMP1 (B) in HeLa S3 Flp-In CTLA4-HA cells transfected for 72 h with non-targeting (NT1) or USP8 siRNA. Scale bar = 15 µm (main figure) or 5 µm (inset). (C and D) Colocalization analysis of CTLA4-HA with EEA1 and LAMP1 in HeLa S3 Flp-In CTLA4-HA for data represented in A and B. Shown are the Pearson’s (C) or Mander’s coefficients (D). Error bars show SD from three independent, color-coded experiments. Opaque circles with black outlines correspond to the mean value from each experiment. Unpaired t test. *P < 0.05, ***P < 0.001. (E and F) Representative confocal images of CTLA4 co-stained for EEA1 (E) or LAMP1 (F) in A2058 cells transfected for 72 h with NT1 or USP8 siRNA. Scale bar = 15 µm (main figure) or 5 µm (inset). (G and H) Colocalization analysis of CTLA4 with EEA1 and LAMP1 in A2058 cells for data represented in E and F. Graphs show Pearson’s (G) or Mander’s coefficients (H) between CTLA4 and EEA1 or LAMP1. Error bars show SD from three independent, color-coded experiments. Opaque circles with black outlines correspond to the mean value from each experiment. Unpaired t test. *P < 0.05, **P < 0.01.

Article Snippet: Human CTLA4-HA DNA flanked by BglII/EcoRI sites was synthesized and provided in a pTWIST-High-Kan vector by TWIST Bioscience, and subcloned into pcDNA3.1 and pEF5/FRT plasmids.

Techniques: Staining, Transfection

Journal: The Journal of Cell Biology

Article Title: Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

doi: 10.1083/jcb.202312141

Figure Lengend Snippet: Regulation of CTLA4 ubiquitylation by USP8 is dependent on HD-PTP. (A) Representative western blots of USP8 co-immunoprecipitated with CTLA4-HA. HeLa S3 Flp-In parental (Par) or CTLA4-HA (HA) cells were transfected for 72 h with non-targeting (NT1), USP8 or HD-PTP siRNA. Cells were lysed and lysates were subjected to HA-immunoprecipitation (IP; IB: Immunoblot). (B) Representative western blots of ubiquitylated CTLA4-HA immunoprecipitated under denaturing conditions. HeLa S3 Flp-In parental (Par) or CTLA4-HA (HA) cells were transfected as in A. Cells were lysed in denaturing SDS lysis buffer and lysates were subjected to immunoprecipitation (IP) with anti-HA coupled magnetic beads. *Antibody heavy chain; ** antibody light chain. (C) Quantification of CTLA4-HA ubiquitylation relative to NT1 for data represented in B. Ubiquitylated CTLA4-HA was normalised to total CTLA4-HA pulled down. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (D) Representative western blots of TUBES pulldown of ubiquitylated CTLA4. HeLa S3 Flp-In CTLA4-HA and A2058 cells were transfected as in A and B and cell lysates subjected to TUBES pulldown. (E) Quantification of CTLA4-HA ubiquitylation relative to NT1 for data represented in D. Ubiquitylated CTLA4(-HA) was normalized to total ubiquitin pulled down. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (F) Representative western blots of CHX chase in HeLa S3 Flp-In CTLA4-HA and A2058 cells following transfection with non-targeting (NT1) or HD-PTP siRNA. Cells were treated with CHX for indicated times before lysis. (G) The half-life of CTLA4 was estimated using an exponential decay model. Error bars indicate SD from three independent experiments. Source data are available for this figure: .

Article Snippet: Human CTLA4-HA DNA flanked by BglII/EcoRI sites was synthesized and provided in a pTWIST-High-Kan vector by TWIST Bioscience, and subcloned into pcDNA3.1 and pEF5/FRT plasmids.

Techniques: Western Blot, Immunoprecipitation, Transfection, Lysis, Magnetic Beads, Ubiquitin Proteomics

Journal: The Journal of Cell Biology

Article Title: Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

doi: 10.1083/jcb.202312141

Figure Lengend Snippet: Catalytic activity and endosomal localization of USP8 are essential for CTLA4 degradation and ubiquitylation. (A) Representative confocal images of HeLa S3 Flp-In CTLA4-HA transfected for 72 h with NT1 or USP8 siRNAs before Cycloheximide (CHX) treatment for indicated times. Cells were fixed and stained for HA. Scale bar = 15 µm. (B) GFP-tagged USP8 siRNA-resistant (USP8*) constructs used in this study. MIT, Microtubule interacting; SBD: SH3 domain binding motif; RHOD, Rhodanese homology domain; USP, Ubiquitin specific protease - catalytic domain. (C) Representative confocal images of HeLa S3 Flp-In CTLA4-HA cells transfected with non-targeting (NT1) or USP8 siRNA and GFP, siRNA-resistant GFP-tagged USP8 (USP8*), catalytically inactive USP8 (C786S*) or ∆MIT-USP8. Cells were treated for 2 h with CHX prior to fixation and staining for HA. Scale bar = 15 µm. (D) Quantification of cells showing rescued phenotypes calculated for data represented in C. Individual data points from 3 (∆MIT), 6 (C786S), or 7 (GFP and USP8*) independent, color-coded experiments are shown. Error bars indicate the SD. Total number of cells analyzed: GFP (599); USP8* (666); C786S (486); ∆MIT (322). One-way ANOVA and Dunnett’s multiple comparison test, ***P < 0.001 and ****P < 0.0001. (E) Representative western blots of ubiquitylated CTLA4-HA immunoprecipitated under denaturing conditions. HeLa S3 Flp-In parental (Par) or CTLA4-HA (HA) cells were transfected with NT1 or USP8 siRNA and either GFP or GFP-tagged siRNA-resistant USP8 constructs as in C. Cells were lysed in denaturing SDS lysis buffer and lysates were subjected to immunoprecipitation (IP) with anti-HA coupled magnetic beads. *Antibody heavy chain; ** antibody light chain. IB: Immunoblot. ( F) Quantification of CTLA4-HA ubiquitylation relative to NT1 for data represented in E. Ubiquitylated CTLA4-HA was normalized to total CTLA4-HA pulled down. Individual data points from two (∆MIT) or five independent, color-coded experiments are shown. Error bars indicate the SD ( n = 5) or range ( n = 2). One-way ANOVA and Dunnett’s multiple comparison test,****P < 0.0001. Source data are available for this figure: .

Article Snippet: Human CTLA4-HA DNA flanked by BglII/EcoRI sites was synthesized and provided in a pTWIST-High-Kan vector by TWIST Bioscience, and subcloned into pcDNA3.1 and pEF5/FRT plasmids.

Techniques: Activity Assay, Transfection, Staining, Construct, Binding Assay, Ubiquitin Proteomics, Comparison, Western Blot, Immunoprecipitation, Lysis, Magnetic Beads

Journal: The Journal of Cell Biology

Article Title: Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

doi: 10.1083/jcb.202312141

Figure Lengend Snippet: Ubiquitylation of CTLA4 on Lys203 and Lys213 is responsible for its rapid turnover. (A) Depiction of the cytoplasmic tail of CTLA4 and the lysine mutants analyzed in this study. (B) Representative western blots of ubiquitylated CTLA4-HA immunoprecipitated under denaturing conditions. HeLa S3 Flp-In parental (Par) or CTLA4-HA WT or lysine mutant cells were transfected for 72 h with non-targeting (NT1) or USP8 siRNA. Cells were lysed in denaturing SDS lysis buffer and lysates were subjected to immunoprecipitation (IP) with anti-HA coupled magnetic beads. *Antibody heavy chain; ** antibody light chain. IB: Immunoblot. (C) Quantification of ubiquitylated lysine mutant CTLA4-HA relative to WT for data represented in B. Ubiquitylated CTLA4-HA is shown normalized to immunoprecipitated CTLA4-HA. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (D) Representative western blots of HeLa S3 Flp-In CTLA4-HA WT, K-null, and K203R,K213R double lysine mutant cells treated with CHX for indicated times before lysis. (E) Quantification of CTLA4-HA turnover for data represented in D. The half-life of CTLA4-HA was estimated using an exponential decay model. Error bars indicate SD from three independent experiments. Source data are available for this figure: .

Article Snippet: Human CTLA4-HA DNA flanked by BglII/EcoRI sites was synthesized and provided in a pTWIST-High-Kan vector by TWIST Bioscience, and subcloned into pcDNA3.1 and pEF5/FRT plasmids.

Techniques: Western Blot, Immunoprecipitation, Mutagenesis, Transfection, Lysis, Magnetic Beads

Journal: The Journal of Cell Biology

Article Title: Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

doi: 10.1083/jcb.202312141

Figure Lengend Snippet: Mutation of K203 and K213 delays CTLA4 turnover. (A) Representative western blots showing increased stability of CTLA4-HA K-null and K203R, K213R mutants. HeLa S3 Flp-In CTLA4-HA WT and indicated lysine mutants were treated with Cycloheximide (CHX) for 2 h before lysis. (B) Quantification of CTLA4-HA remaining following CHX treatment. Individual data points from three independent, color-coded experiments are shown. Error bars show SD. (C) Representative western blots of HeLa S3 Flp-In CTLA4-HA WT, K-null and K203R, K213R double lysine mutants treated for 4 h with Cycloheximide (CHX) alone or together with Concanamycin A (ConcA) or Epoxomicin (Epo) prior to lysis. (D) Quantification of CTLA4-HA remaining normalised to control treated cells for data represented in C. Individual data points from three independent, color-coded experiments are shown. Error bars show SD. Two-way ANOVA multiple comparisons with uncorrected Fisher’s LSD, *P < 0.05, **P < 0.01. (E and F) Representative confocal images of CTLA4-HA WT, K-null, and K203R,K213R mutants co-stained with EEA1 (E) or LAMP1 (F). Scale bar = 15 µm (main figure) and 5 µm (inset). (G and H) Co-localization analysis of CTLA4-HA WT, K-null and K203R,K213R, and with EEA1 (G) and LAMP1 (H). Graphs show Pearson’s coefficients or Mander’s coefficients. Error bars indicate SD for three independent, color-coded experiments. Opaque circles with dark outlines correspond to the mean value from each experiment. One-way ANOVA with Dunnett’s multiple comparisons test, *P < 0.05, **P < 0.01. Source data are available for this figure: .

Article Snippet: Human CTLA4-HA DNA flanked by BglII/EcoRI sites was synthesized and provided in a pTWIST-High-Kan vector by TWIST Bioscience, and subcloned into pcDNA3.1 and pEF5/FRT plasmids.

Techniques: Mutagenesis, Western Blot, Lysis, Control, Staining

Journal: The Journal of Cell Biology

Article Title: Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

doi: 10.1083/jcb.202312141

Figure Lengend Snippet: Ubiquitin chain restriction (UbiCRest) analysis reveals K63, K27, and K29 ubiquitin chain association with CTLA4-HA. (A) Representative UbiCRest analysis of western blots for CTLA4-HA. HeLa S3 Flp-In CTLA4-HA were transfected for 72 h with non-targeting (NT1) or USP8 siRNA prior to lysis and HA-immunoprecipitation (IP; IB: Immunoblot). CTLA4-HA-beads were treated with indicated DUBs or buffer only (Mock) for 1 h at 37°C, and analyzed alongside supernatants (released ubiquitin species). Specific activities of DUBs as reported in the literature are depicted in the key above the blot. Ub-P indicates the ability to cleave proximal ubiquitin; S/T denotes activity for serine or threonine linkages. All l.s.: All linkage-specific DUBs (OTULIN, OTUB1*, AMSH*, LotA N , Cezanne, TRABID, OTUD2, TssM*). (B) Quantification of Ub-CTLA4-HA signal remaining after DUB-treatment shown relative to Mock (“buffer only” control) for data represented in A. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (C) Representative western blots of CTLA4-HA immunoprecipitated (IP) under denaturing conditions were probed with K63 (top), K27 (middle), and K29 (bottom) ubiquitin chain linkage-specific antibodies. (D) Left Panel: A balance of E3 ligase and deubiquitylating activity governs receptor fate at the endosome. Endosomal USP8 is proposed to recycle ubiquitin from CTLA4 prior as well as after commitment to the lysosomal degradation pathway. Middle Panel: In the absence of USP8, a complex pattern of ubiquitylation accrues, composed of conventional Lys63 and unusual Lys27 and Lys29 ubiquitin linkages. This would normally lead to rapid degradation but is countermanded by downstream effects of USP8 loss that inhibit delivery to a degradative lysosome. That some ubiquitylated CTLA4 reaches the internal vesicles of the multivesicular body can be inferred from its presence in exosomes released upon v-ATPase inhibition. Right panel: Mutation of all lysines (K-null) or just the critical two lysines (K203 and K213) to arginines in the cytoplasmic tail of CTLA4 interferes with endolysosomal sorting and downregulation. These CTLA4 mutants accumulate in early (sorting) endosomes and fail to progress to later endolysosomal compartments. For reasons of clarity, mono-ubiquitylation, branching, and diversity of chain length are not depicted. Source data are available for this figure: .

Article Snippet: Human CTLA4-HA DNA flanked by BglII/EcoRI sites was synthesized and provided in a pTWIST-High-Kan vector by TWIST Bioscience, and subcloned into pcDNA3.1 and pEF5/FRT plasmids.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Lysis, Immunoprecipitation, Activity Assay, Control, Inhibition, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation

doi: 10.1083/jcb.202312141

Figure Lengend Snippet: Combinatorial UbiCRest analysis of CTLA4-HA. (A) Line graphs corresponding to the Ub-CTLA4-HA signal shown in . (B) Quantification of total ubiquitin released into the supernatant by DUBs relative to USP2 for data represented in . Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (C and D) Representative western blot showing K29- (C) and K27-linked (D) ubiquitin associated with immunoisolated CTLA4-HA after UbiCRest treatment with indicated DUBs. HeLa S3 Flp-In CTLA4-HA cells were transfected for 72 h with USP8 siRNA prior to lysis. CTLA4-HA was immunoprecipitated using anti-HA magnetic beads and incubated for 1 h at 37°C with the indicated DUBs. (E) Representative western blot of UbiCRest analysis of CTLA4 using a combination of linkage-specific DUBs. HeLa S3 Flp-In CTLA4-HA cells were transfected for 72 h with NT1 or USP8 siRNA prior to lysis. CTLA4-HA was immunoprecipitated using anti-HA magnetic beads and incubated for 1 h at 37°C with the indicated DUBs. Supernatants containing ubiquitin species released by DUBs were collected and analyzed in parallel with CTLA4-HA eluted from the beads. Specific activities of DUBs as reported in the literature are depicted in the key above the blot. Ub-P indicates ability to cleave proximal ubiquitin. Source data are available for this figure: .

Article Snippet: Human CTLA4-HA DNA flanked by BglII/EcoRI sites was synthesized and provided in a pTWIST-High-Kan vector by TWIST Bioscience, and subcloned into pcDNA3.1 and pEF5/FRT plasmids.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Lysis, Immunoprecipitation, Magnetic Beads, Incubation

Journal: ACS synthetic biology

Article Title: Streamlining the Design-to-Build Transition with Build-Optimization Software Tools.

doi: 10.1021/acssynbio.6b00200

Figure Lengend Snippet: Figure 2 shows screenshots of the BOOST Web UI, and the organization of the screenshots provides a process-oriented walkthrough of the BOOST Juggler, Polisher and Partitioner tools that respectively automate the tasks of 'Codon Adjustment', 'Sequence Polishing', and 'Sequence Partitioning' of the design for DNA synthesis and assembly workflow (see Figure 1). As shown in Figure 2 the output sequences of one tool serve as the input of the next downstream tool. In all three BOOST tools, the user can enter either single sequences or a batch of sequences in CSV, Fasta, Genbank, or SBOL file format. Every tool automatically detects the file format and the sequence type. Since any DNA sequence can theoretically be a protein sequence, the user can overrule the detected sequence type. Both the BOOST Juggler and the BOOST Polisher tool prefer annotated input sequences, such as provided by the Genbank and SBOL sequence

Article Snippet: In Table 1 we compare the BOOST functionalities against the functionalities provided on the web portals of commercial DNA synthesis providers (IDT, Gen9, ThermoFisher, Twist, and DNA2.0) and DNA design tools (DNA2.0’s GeneDesigner34, GeneDesign18, GenomeCalligrapher35).

Techniques: Sequencing, DNA Synthesis

Journal: ACS synthetic biology

Article Title: Streamlining the Design-to-Build Transition with Build-Optimization Software Tools.

doi: 10.1021/acssynbio.6b00200

Figure Lengend Snippet: Figure 3: An example of verifying a DNA sequence against gene synthesis constraints and reporting violations in a comprehensible manner.

Article Snippet: In Table 1 we compare the BOOST functionalities against the functionalities provided on the web portals of commercial DNA synthesis providers (IDT, Gen9, ThermoFisher, Twist, and DNA2.0) and DNA design tools (DNA2.0’s GeneDesigner34, GeneDesign18, GenomeCalligrapher35).

Techniques: Sequencing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: N-Acetyltransferase 10 Enhances Doxorubicin Resistance in Human Hepatocellular Carcinoma Cell Lines by Promoting the Epithelial-to-Mesenchymal Transition

doi: 10.1155/2019/7561879

Figure Lengend Snippet: Inhibition of NAT10 using remodelin increases the chemosensitivity of HCC cell lines to doxorubicin. (a–d) CCK-8 assay of cell viability. (e–h) Representative images and quantification of EdU incorporation assay of cell growth and DNA synthesis. ∗ P < 0.05, doxorubicin vs. control cells; # P < 0.05, doxorubicin+remodelin vs. remodelin.

Article Snippet: The NAT10 siRNA (sc62660) and Twist siRNA (sc38604) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Inhibition, CCK-8 Assay, DNA Synthesis, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: N-Acetyltransferase 10 Enhances Doxorubicin Resistance in Human Hepatocellular Carcinoma Cell Lines by Promoting the Epithelial-to-Mesenchymal Transition

doi: 10.1155/2019/7561879

Figure Lengend Snippet: Knocking down and inhibiting NAT10 increase the chemosensitivity of HCC cell lines to doxorubicin. (a) Western blotting confirmation of NAT10 knockdown efficiency in siRNA-transfected cells. (b–e) CCK-8 assay of the viability of HCC cells treated with doxorubicin, the NAT10 siRNA, or doxorubicin and remodelin following knockdown of NAT10.

Article Snippet: The NAT10 siRNA (sc62660) and Twist siRNA (sc38604) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Western Blot, Knockdown, Transfection, CCK-8 Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: N-Acetyltransferase 10 Enhances Doxorubicin Resistance in Human Hepatocellular Carcinoma Cell Lines by Promoting the Epithelial-to-Mesenchymal Transition

doi: 10.1155/2019/7561879

Figure Lengend Snippet: Inhibition of NAT10 using remodelin reverses the EMT in HCC cell lines. (a) Western blotting of NAT10, E-cadherin, and vimentin expression; ∗ P < 0.05. (b) Immunofluorescence analysis of E-cadherin and vimentin expression in cells treated with or without remodelin (IC 50 of remodelin in combination).

Article Snippet: The NAT10 siRNA (sc62660) and Twist siRNA (sc38604) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Inhibition, Western Blot, Expressing, Immunofluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: N-Acetyltransferase 10 Enhances Doxorubicin Resistance in Human Hepatocellular Carcinoma Cell Lines by Promoting the Epithelial-to-Mesenchymal Transition

doi: 10.1155/2019/7561879

Figure Lengend Snippet: Inhibition of NAT10 reverses the doxorubicin-induced EMT in HCC cell lines. Western blotting and immunofluorescence analysis of the expression of EMT-related markers E-cadherin and vimentin in cells treated with doxorubicin or remodelin (a, b) or HCC cells transfected with the NAT10 siRNA (c).

Article Snippet: The NAT10 siRNA (sc62660) and Twist siRNA (sc38604) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Inhibition, Western Blot, Immunofluorescence, Expressing, Transfection

Journal: Oxidative Medicine and Cellular Longevity

Article Title: N-Acetyltransferase 10 Enhances Doxorubicin Resistance in Human Hepatocellular Carcinoma Cell Lines by Promoting the Epithelial-to-Mesenchymal Transition

doi: 10.1155/2019/7561879

Figure Lengend Snippet: Inhibition of NAT10 reverses the hypoxia-induced EMT in HCC cell lines. (a, b) CCK-8 assay of Huh-7 and BEL-7402 cell viability treated with doxorubicin, remodelin, or doxorubicin plus remodelin under hypoxic conditions. (c) Western blotting analysis of E-cadherin and vimentin expression in Huh-7 and BEL-7402 cells subjected to hypoxic conditions. (c) Immunofluorescence analysis of E-cadherin and vimentin expression.

Article Snippet: The NAT10 siRNA (sc62660) and Twist siRNA (sc38604) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Inhibition, CCK-8 Assay, Western Blot, Expressing, Immunofluorescence