Journal: Journal of Biological Chemistry

Article Title: Degradation of the endoplasmic reticulum–anchored transcription factor MyRF by the ubiquitin ligase SCFFbxw7 in a manner dependent on the kinase GSK-3

doi: 10.1074/jbc.ra117.000741

Figure Lengend Snippet: FIGURE 2. SCFFbxw7α ubiquitylates MyRF. A, Co-immunoprecipitation of HA-tagged MyRF as well as of Cul1, Skp1, and c-Myc with FLAG-tagged WT or ΔF mutant forms of Fbxw7α from mHepa cell lysates. Immunoprecipitates (IP) prepared with antibodies to FLAG from cells expressing the recombinant Fbxw7α proteins (or from those transfected with the corresponding empty vector, Mock), as well as the original cell lysates (Input), were subjected to immunoblot (IB) analysis with antibodies to HA, to FLAG, or to the indicated proteins. Hsp90 was examined as a loading control. FL, N, and the asterisk indicate full-length and nuclear forms of MyRF as well as a nonspecific band, respectively. B, Cycloheximide chase analysis of MyRF in mHepa cells stably expressing HA-tagged MyRF with or without Fbxw7α. Cells incubated with cycloheximide (CHX, 100 µg/ml) for the indicated times were subjected to immunoblot analysis with antibodies to HA. C, The percentage of HA-tagged MyRF(N) remaining after the various chase times in B was quantitated with ImageJ software. D, HA-tagged MyRF(N) (amino acids 1–586 of MyRF) was subjected to an in vitro ubiquitylation assay with immunopurified SCFFbxw7α and ATP as well as with E1, E2, and ubiquitin (Ub), as indicated. Reaction mixtures (10 µl) were subjected to immunoblot analysis with antibodies to HA. The positions of unmodified and polyubiquitylated (Ubn) forms of MyRF(N) are indicated.

Article Snippet: Antibodies—Antibodies to the FLAG epitope (M2) were obtained from Sigma (St. Louis, MO); those to the HA epitope (HA.11 or Y-11) from Covance (Princeton, NJ) or Santa Cruz Biotechnology (Dallas, TX), respectively; those to Skp1, to Hsp90, and to CD140a (PDGFRα) from BD Biosciences (San Jose, CA); those to Cul1 from Cell Signaling Technology (Danvers, MA); and those to c-Myc from Abcam (Cambridge, UK).

Techniques: Immunoprecipitation, Mutagenesis, Expressing, Recombinant, Transfection, Plasmid Preparation, Western Blot, Control, Stable Transfection, Incubation, Software, In Vitro, Ubiquitin Assay, Ubiquitin Proteomics

Journal: iScience

Article Title: O -GlcNAcylation regulates OTX2's proteostasis.

doi: 10.1016/j.isci.2023.108184

Figure Lengend Snippet: Figure 1. OTX2 is primarily modified in its central retention domain (A) Electron-transfer/higher-energy collision dissociation–mass spectrometry (EThcD-MS) spectrum of the m/z 759.0495 3+ precursor of an OTX2 peptide modified by HexNAc on serine 135 (lowercase). (B) Table summary of modified peptides. Stars indicate O-GlcNAc sites identified by mass spectrometry. HD, homeodomain; RD, Retention Domain; TF, transcription factor domain; OT, transactivation domain; NRS, nuclear retention sequence; SIWSPAS, corepressor interaction motif. AlphaFold protein structure of OTX2 annotated with the O-GlcNAc sites identified (blue spheres), O-GlcNAcylated peptides with no specific residue identified (blue ribbon), and the homeodomain (maroon). (C) WGA pulldown of outlined constructs in HeLa cells. FL, Myc-tagged OTX2 full length; DRD, Retention Domain deletion; RD, Retention Domain only. Western blot (WB) bands were quantified as unbound (Non-O-GlcNAc) and WGA-bound (O-GlcNAc) and normalized to the amount of each construct in the total extract WB. (D) Immunoblot of pulldowns of OTX2 O-GlcNAc site mutants and WT OTX2 transfected into HeLa cells. (E) Quantification by the optic density of representative blot (D) and biological replicates. One-way ANOVA, p < 0.05 (*), p < 0.01 (**) (n = 3). (F) HeLa cells transfected with WT or 3A mutant OTX2. Phosphorylated species were visualized by PhosTag SDS-PAGE. (G) Optical density quantification (n = 3). Data are represented as mean G SEM in panels presenting pooled data. See also Figures S1, S2, Tables S1, and S2.

Article Snippet: Antibodies Rabbit polyclonal anti-OTX2 Proteintech Cat# 13497-1-AP; RRID:AB_2157176 Mouse monoclonal anti-O-Linked N-Acetylglucosamine antibody (RL2) Abcam Cat# ab2739; RRID:AB_303264 Rabbit polyclonal anti-Actin Millipore Sigma Cat# A2066; RRID:AB_476693 Mouse monoclonal anti-b-Actin Millipore Sigma Cat# A5441; RRID:AB_476744 Mouse monoclonal anti-Ubiquitin (eBioP4D1 (P4D1)) Thermo Fisher Scientific Cat# 14-6078-82; RRID:AB_837154 Rabbit polyclonal anti-LC3B Proteintech Cat# 18725-1-AP; RRID:AB_2137745 Rabbit polyclonal anti-c-Myc Millipore Sigma Cat# C3956; RRID:AB_439680 Mouse monoclonal anti-DYKDDDDK (FLAG) Tag (FG4R) Thermo Fisher Scientific Cat# MA1-91878; RRID:AB_1957945 Mouse monoclonal anti-alpha-Tubulin (961258) Novus Biologicals Cat# MAB93441; RRID:AB_2938603 Rabbit polyclonal anti-Lamin A/C Novus Biologicals Cat# NB100-56649; RRID:AB_838524 Mouse monoclonal anti-GAPDH Abcam Cat# ab9484; RRID:AB_307274 Rabbit polyclonal anti-CCT5 Bethyl Laboratories Cat# A303-481A; RRID:AB_10952578 Rabbit polyclonal anti-SQSTM1/p62 Abcam Cat# ab91526; RRID:AB_2050336 Bacterial and virus strains One Shot BL21 Star (DE3) Chemically Competent E. coli cells Invitrogen Cat# C601003 Chemicals, peptides, and recombinant proteins Thiamet G Millipore Sigma Cat# SML0244 Chloroquine diphosphate salt Chem Impex Cat# 22113 MG-132 Millipore Sigma Cat# 474790 Bortezomib Millipore Sigma Cat# 504314 Cycloheximide Millipore Sigma Cat# 01810 PUGNAc Millipore Sigma Cat# A7229 Doxycycline TCI Chemicals Cat# D4116 OSMI4 MedChemExpress Cat# HY-114361 DRB Millipore Sigma Cat# D1916 Rapamycin Cell Signaling Cat# 9904 DSP Pierce Cat# A35393 Phos binding reagent acrylamide ApexBio Cat# F4002 Lambda Protein Phosphatase New England BioLabs Cat# P0753S Critical commercial assays In-Fusion Snap assembly Takara Cat# 638947 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies Cat# 200523

Techniques: Mass Spectrometry, Sequencing, Residue, Construct, Western Blot, Transfection, Mutagenesis, SDS Page

Journal: iScience

Article Title: O -GlcNAcylation regulates OTX2's proteostasis.

doi: 10.1016/j.isci.2023.108184

Figure Lengend Snippet: Figure 2. O-GlcNAc prevents OTX2 macroautophagy-dependent degradation (A) HeLa cells transfected with WT OTX2 or O-GlcNAc deficient mutants S136A, T137A, or 3A (S135A/S136A/T137A), treated with cycloheximide (CHX) or vehicle for up to 5 h and analyzed by Western Blot (WB). (B) Optical density (OD) quantification of individual site mutants S136A and T137A. Mutant vs. WT, mixed-effects analysis, p < 0.05 (*) (n = 3). (C) OD quantification of 3A mutant vs. WT, two-way ANOVA, p < 0.05 (*), p < 0.01 (**) (n = 3). (D) OTX2 degradation timeline upon Doxycycline (DOX)-triggered OTX2 KD or CHX treatment for A6#20 human medulloblastoma cells, or CHX treatment for HeLa cells transfected with WT-OTX2. Half-lives were calculated by non-linear regression analysis. (E) WB of A6#20 cells treated with DOX, MG132, chloroquine (CQ), or vehicle, for up to 20 h. (F) OD quantification of WB (n = 3). Two-way ANOVA, DOX vs. DOX+MG, p < 0.05 (#); DOX vs. DOX+MG+CQ, p < 0.01 (**), p < 0.001 (***). (G) A6#20 cells transfected with WT OTX2 were treated with CHX, MG132, CQ, or vehicle overnight. (H) A6#20 cells transfected with WT OTX2 were treated with MG132, CQ, or vehicles for 2 h. (I) Western blot of A6#20 medulloblastoma cells treated overnight with MG132, chloroquine (CQ), Thiamet-G (TG), and doxycycline (DOX), individually and in combination. (J) Quantification of Western blot (I) and biological replicates. One-way ANOVA, p < 0.05 (*), p < 0.01 (**) (n = 5). (K and L) HeLa cells transfected with WT (K) or 3A-OTX2 (L) and treated with CHX supplemented with CQ (n = 2), two-way ANOVA, p < 0.01 (**), p < 0.001 (***). Data are represented as mean G SEM in panels presenting pooled data. See also Figures S3 and S4.

Article Snippet: Antibodies Rabbit polyclonal anti-OTX2 Proteintech Cat# 13497-1-AP; RRID:AB_2157176 Mouse monoclonal anti-O-Linked N-Acetylglucosamine antibody (RL2) Abcam Cat# ab2739; RRID:AB_303264 Rabbit polyclonal anti-Actin Millipore Sigma Cat# A2066; RRID:AB_476693 Mouse monoclonal anti-b-Actin Millipore Sigma Cat# A5441; RRID:AB_476744 Mouse monoclonal anti-Ubiquitin (eBioP4D1 (P4D1)) Thermo Fisher Scientific Cat# 14-6078-82; RRID:AB_837154 Rabbit polyclonal anti-LC3B Proteintech Cat# 18725-1-AP; RRID:AB_2137745 Rabbit polyclonal anti-c-Myc Millipore Sigma Cat# C3956; RRID:AB_439680 Mouse monoclonal anti-DYKDDDDK (FLAG) Tag (FG4R) Thermo Fisher Scientific Cat# MA1-91878; RRID:AB_1957945 Mouse monoclonal anti-alpha-Tubulin (961258) Novus Biologicals Cat# MAB93441; RRID:AB_2938603 Rabbit polyclonal anti-Lamin A/C Novus Biologicals Cat# NB100-56649; RRID:AB_838524 Mouse monoclonal anti-GAPDH Abcam Cat# ab9484; RRID:AB_307274 Rabbit polyclonal anti-CCT5 Bethyl Laboratories Cat# A303-481A; RRID:AB_10952578 Rabbit polyclonal anti-SQSTM1/p62 Abcam Cat# ab91526; RRID:AB_2050336 Bacterial and virus strains One Shot BL21 Star (DE3) Chemically Competent E. coli cells Invitrogen Cat# C601003 Chemicals, peptides, and recombinant proteins Thiamet G Millipore Sigma Cat# SML0244 Chloroquine diphosphate salt Chem Impex Cat# 22113 MG-132 Millipore Sigma Cat# 474790 Bortezomib Millipore Sigma Cat# 504314 Cycloheximide Millipore Sigma Cat# 01810 PUGNAc Millipore Sigma Cat# A7229 Doxycycline TCI Chemicals Cat# D4116 OSMI4 MedChemExpress Cat# HY-114361 DRB Millipore Sigma Cat# D1916 Rapamycin Cell Signaling Cat# 9904 DSP Pierce Cat# A35393 Phos binding reagent acrylamide ApexBio Cat# F4002 Lambda Protein Phosphatase New England BioLabs Cat# P0753S Critical commercial assays In-Fusion Snap assembly Takara Cat# 638947 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies Cat# 200523

Techniques: Transfection, Western Blot, Mutagenesis

Journal: iScience

Article Title: O -GlcNAcylation regulates OTX2's proteostasis.

doi: 10.1016/j.isci.2023.108184

Figure Lengend Snippet: Figure 3. OTX2 aggregation is regulated by O-GlcNAcylation (A) A6#20 cells transfected with WT OTX2 and resolved on SDS-PAGE in denaturing or non-denaturing conditions. (B) A6#20 cells treated overnight with MG132, chloroquine (CQ), or vehicle and crosslinked with DSP. Samples in either reducing (b-mercaptoethanol, +BME) or non-reducing (-BME) sample buffer were analyzed by WB. (C and D) HeLa cells were transfected with either WT OTX2 or empty vector (EV-OSF) and treated overnight with the O-GlcNAc inhibitors OSMI4, Thiamet-G (TG), or DMSO vehicle. Proteins were crosslinked with DSP at 100 mM (C) or 1 mM (D), lysates separated by soluble and pellet fractions. All samples were run under reducing conditions (+BME), (n = 3), one-way ANOVA, p < 0.05 (*). In (D), OTX2 was pulled down with Strep-Tactin beads. Pie charts graph the OD quantification of WB bands in each fraction for each treatment condition. (E) HeLa cells were transfected with WT or mutant 3A-OTX2. Cells were crosslinked with DSP (100 mM) before harvest, and the soluble and pellet lysates were run under reducing conditions (+BME). Student’s t test, (n = 2). Data are represented as mean G SEM in panels presenting pooled data. See also Figures S5A–S5F.

Article Snippet: Antibodies Rabbit polyclonal anti-OTX2 Proteintech Cat# 13497-1-AP; RRID:AB_2157176 Mouse monoclonal anti-O-Linked N-Acetylglucosamine antibody (RL2) Abcam Cat# ab2739; RRID:AB_303264 Rabbit polyclonal anti-Actin Millipore Sigma Cat# A2066; RRID:AB_476693 Mouse monoclonal anti-b-Actin Millipore Sigma Cat# A5441; RRID:AB_476744 Mouse monoclonal anti-Ubiquitin (eBioP4D1 (P4D1)) Thermo Fisher Scientific Cat# 14-6078-82; RRID:AB_837154 Rabbit polyclonal anti-LC3B Proteintech Cat# 18725-1-AP; RRID:AB_2137745 Rabbit polyclonal anti-c-Myc Millipore Sigma Cat# C3956; RRID:AB_439680 Mouse monoclonal anti-DYKDDDDK (FLAG) Tag (FG4R) Thermo Fisher Scientific Cat# MA1-91878; RRID:AB_1957945 Mouse monoclonal anti-alpha-Tubulin (961258) Novus Biologicals Cat# MAB93441; RRID:AB_2938603 Rabbit polyclonal anti-Lamin A/C Novus Biologicals Cat# NB100-56649; RRID:AB_838524 Mouse monoclonal anti-GAPDH Abcam Cat# ab9484; RRID:AB_307274 Rabbit polyclonal anti-CCT5 Bethyl Laboratories Cat# A303-481A; RRID:AB_10952578 Rabbit polyclonal anti-SQSTM1/p62 Abcam Cat# ab91526; RRID:AB_2050336 Bacterial and virus strains One Shot BL21 Star (DE3) Chemically Competent E. coli cells Invitrogen Cat# C601003 Chemicals, peptides, and recombinant proteins Thiamet G Millipore Sigma Cat# SML0244 Chloroquine diphosphate salt Chem Impex Cat# 22113 MG-132 Millipore Sigma Cat# 474790 Bortezomib Millipore Sigma Cat# 504314 Cycloheximide Millipore Sigma Cat# 01810 PUGNAc Millipore Sigma Cat# A7229 Doxycycline TCI Chemicals Cat# D4116 OSMI4 MedChemExpress Cat# HY-114361 DRB Millipore Sigma Cat# D1916 Rapamycin Cell Signaling Cat# 9904 DSP Pierce Cat# A35393 Phos binding reagent acrylamide ApexBio Cat# F4002 Lambda Protein Phosphatase New England BioLabs Cat# P0753S Critical commercial assays In-Fusion Snap assembly Takara Cat# 638947 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies Cat# 200523

Techniques: Transfection, SDS Page, Plasmid Preparation, Mutagenesis

Journal: iScience

Article Title: O -GlcNAcylation regulates OTX2's proteostasis.

doi: 10.1016/j.isci.2023.108184

Figure Lengend Snippet: Figure 4. OTX2 O-GlcNAc-site mutants lose interactions with the chaperonin CCT5 (A) Volcano plot of proteins commonly lost or gained as OTX2 interactors in WT and mutant constructs (n = 2). (B) STRING network analysis of the 19 common proteins dislodged from OTX2 when one or all O-GlcNAc sites found were mutated to alanine. (C) Interactome analysis of the TriC complex. (D) HeLa cells transfected with OTX2 WT or mutant 3A. OTX2 was pulled down and analyzed by Western Blot. Optical density quantification of Western Blot bands. One-way ANOVA, p < 0.05 (*) (n = 2). (E) HeLa cells transfected with OTX2 WT or empty vector EV-OSF, lysates were crosslinked with DSP. OTX2 was pulled down by Strep-Tactin beads from both soluble and pellet fractions and analyzed by Western Blot. Data are represented as mean G SEM in panels presenting pooled data. See also Figures S5G, S5H and Table S3.

Article Snippet: Antibodies Rabbit polyclonal anti-OTX2 Proteintech Cat# 13497-1-AP; RRID:AB_2157176 Mouse monoclonal anti-O-Linked N-Acetylglucosamine antibody (RL2) Abcam Cat# ab2739; RRID:AB_303264 Rabbit polyclonal anti-Actin Millipore Sigma Cat# A2066; RRID:AB_476693 Mouse monoclonal anti-b-Actin Millipore Sigma Cat# A5441; RRID:AB_476744 Mouse monoclonal anti-Ubiquitin (eBioP4D1 (P4D1)) Thermo Fisher Scientific Cat# 14-6078-82; RRID:AB_837154 Rabbit polyclonal anti-LC3B Proteintech Cat# 18725-1-AP; RRID:AB_2137745 Rabbit polyclonal anti-c-Myc Millipore Sigma Cat# C3956; RRID:AB_439680 Mouse monoclonal anti-DYKDDDDK (FLAG) Tag (FG4R) Thermo Fisher Scientific Cat# MA1-91878; RRID:AB_1957945 Mouse monoclonal anti-alpha-Tubulin (961258) Novus Biologicals Cat# MAB93441; RRID:AB_2938603 Rabbit polyclonal anti-Lamin A/C Novus Biologicals Cat# NB100-56649; RRID:AB_838524 Mouse monoclonal anti-GAPDH Abcam Cat# ab9484; RRID:AB_307274 Rabbit polyclonal anti-CCT5 Bethyl Laboratories Cat# A303-481A; RRID:AB_10952578 Rabbit polyclonal anti-SQSTM1/p62 Abcam Cat# ab91526; RRID:AB_2050336 Bacterial and virus strains One Shot BL21 Star (DE3) Chemically Competent E. coli cells Invitrogen Cat# C601003 Chemicals, peptides, and recombinant proteins Thiamet G Millipore Sigma Cat# SML0244 Chloroquine diphosphate salt Chem Impex Cat# 22113 MG-132 Millipore Sigma Cat# 474790 Bortezomib Millipore Sigma Cat# 504314 Cycloheximide Millipore Sigma Cat# 01810 PUGNAc Millipore Sigma Cat# A7229 Doxycycline TCI Chemicals Cat# D4116 OSMI4 MedChemExpress Cat# HY-114361 DRB Millipore Sigma Cat# D1916 Rapamycin Cell Signaling Cat# 9904 DSP Pierce Cat# A35393 Phos binding reagent acrylamide ApexBio Cat# F4002 Lambda Protein Phosphatase New England BioLabs Cat# P0753S Critical commercial assays In-Fusion Snap assembly Takara Cat# 638947 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies Cat# 200523

Techniques: Mutagenesis, Construct, Transfection, Western Blot, Plasmid Preparation

Journal: iScience

Article Title: O -GlcNAcylation regulates OTX2's proteostasis.

doi: 10.1016/j.isci.2023.108184

Figure Lengend Snippet: Figure 5. O-GlcNAc depleted OTX2 overexpression leads to cytotoxicity (A) OTX2 transcriptional activity assay of WT- and 3A-OSF constructs in HeLa cells. Activity measured after treatment with TG, OSMI4, or vehicle (NT) (n = 9). Each value was normalized to their corresponding Empty Vector (EV) treated sample. Welch ANOVA, p < 0.01 (**), p < 0.0001 (****). (B) MTT assay of HeLa cells transfected with OTX2 WT or 3A mutant for up to 48 h. Each time point value was normalized to the corresponding EV sample (EV: n = 3, WT/3A: n = 6). Multiple unpaired t-tests, p < 0.05 (*), p < 0.01 (**), p < 0.0001 (****). (C) MTT assay of HeLa cells transfected with OTX2 WT or mutant 3A and treated with OSMI4, TG, or vehicles (NT) for 24 h (n = 6). Each value was normalized to their corresponding EV treatment sample. Welch ANOVA, p < 0.01 (**), p < 0.0001 (****). (D) Proposed model of OTX2 turnover regulation by O-GlcNAcylation. Created with BioRender.com. Data are represented as mean G SEM in panels presenting pooled data. See also Figure S6.

Article Snippet: Antibodies Rabbit polyclonal anti-OTX2 Proteintech Cat# 13497-1-AP; RRID:AB_2157176 Mouse monoclonal anti-O-Linked N-Acetylglucosamine antibody (RL2) Abcam Cat# ab2739; RRID:AB_303264 Rabbit polyclonal anti-Actin Millipore Sigma Cat# A2066; RRID:AB_476693 Mouse monoclonal anti-b-Actin Millipore Sigma Cat# A5441; RRID:AB_476744 Mouse monoclonal anti-Ubiquitin (eBioP4D1 (P4D1)) Thermo Fisher Scientific Cat# 14-6078-82; RRID:AB_837154 Rabbit polyclonal anti-LC3B Proteintech Cat# 18725-1-AP; RRID:AB_2137745 Rabbit polyclonal anti-c-Myc Millipore Sigma Cat# C3956; RRID:AB_439680 Mouse monoclonal anti-DYKDDDDK (FLAG) Tag (FG4R) Thermo Fisher Scientific Cat# MA1-91878; RRID:AB_1957945 Mouse monoclonal anti-alpha-Tubulin (961258) Novus Biologicals Cat# MAB93441; RRID:AB_2938603 Rabbit polyclonal anti-Lamin A/C Novus Biologicals Cat# NB100-56649; RRID:AB_838524 Mouse monoclonal anti-GAPDH Abcam Cat# ab9484; RRID:AB_307274 Rabbit polyclonal anti-CCT5 Bethyl Laboratories Cat# A303-481A; RRID:AB_10952578 Rabbit polyclonal anti-SQSTM1/p62 Abcam Cat# ab91526; RRID:AB_2050336 Bacterial and virus strains One Shot BL21 Star (DE3) Chemically Competent E. coli cells Invitrogen Cat# C601003 Chemicals, peptides, and recombinant proteins Thiamet G Millipore Sigma Cat# SML0244 Chloroquine diphosphate salt Chem Impex Cat# 22113 MG-132 Millipore Sigma Cat# 474790 Bortezomib Millipore Sigma Cat# 504314 Cycloheximide Millipore Sigma Cat# 01810 PUGNAc Millipore Sigma Cat# A7229 Doxycycline TCI Chemicals Cat# D4116 OSMI4 MedChemExpress Cat# HY-114361 DRB Millipore Sigma Cat# D1916 Rapamycin Cell Signaling Cat# 9904 DSP Pierce Cat# A35393 Phos binding reagent acrylamide ApexBio Cat# F4002 Lambda Protein Phosphatase New England BioLabs Cat# P0753S Critical commercial assays In-Fusion Snap assembly Takara Cat# 638947 QuikChange II Site-Directed Mutagenesis Kit Agilent Technologies Cat# 200523

Techniques: Over Expression, Activity Assay, Construct, Plasmid Preparation, MTT Assay, Transfection, Mutagenesis

Journal: Journal of cell science

Article Title: Tbl1 promotes Wnt-β-catenin signaling-induced degradation of the Tcf7l1 protein in mouse embryonic stem cells.

doi: 10.1242/jcs.261241

Figure Lengend Snippet: Fig. 1. Overexpression of Tbl genes decreases Tcf7l1 protein levels and is beneficial for maintaining the stemness of mESCs. (A) Western blot analysis of Tcf7l1 protein levels in cells overexpressing Flag tag alone (empty vector control, Flag) or Flag–Tbl1. (B) Densitometric analysis of the relative protein levels of Tcf7l1, as shown in A, was performed with ImageJ software. Protein levels were normalized to those of β-actin. (C) Western blot analysis of Tcf7l1 levels in cells overexpressing Flag tag alone (Flag) or Flag–Tblr1. (D) Densitometric analysis of the relative protein level of Tcf7l1, as shown in C, was performed with ImageJ software. Protein levels were normalized to those of β-actin. (E) RT–qPCR analysis of Tcf7l1 levels in cells overexpressing Flag tag alone (Flag) or Flag–Tbl1. (F) RT–qPCR analysis of Tcf7l1 levels in cells overexpressing Flag tag alone (Flag) or Flag–Tblr1. (G) AP staining of mESCs expressing Flag tag alone, Flag–Tbl1 or Flag–Tblr1 cultured in serum-containing medium without LIF for 8 days. Scale bars: 100 μm. (H) Quantification of AP-positive colonies as shown in G. (I) RT–qPCR analysis of the expression of Oct4, Sox2, Klf4, Esrrb, Nanog and Gata4 in mESCs expressing Flag tag alone, Flag–Tbl1 or Flag–Tblr1. (J) Left: AP staining of mESCs expressing either Flag tag alone or Flag–Tbl1 and transfected with a plasmid encoding HA– Tcf7l1. Cells were cultured in medium containing LIF and serum. Right: immunofluorescence (IF) of HA–Tcf7l1 mESCs transfected with Flag or Flag–Tbl1 showing Oct4 (red) and Hoechst 33342 (blue). IF images are representative of three experiments. Scale bars: 100 μm. (K) Quantification of AP-positive colonies as shown in J. All quantitative data are presented as mean±s.d. of n=3 biological replicates. *P<0.05, **P<0.01 versus Flag, as determined by unpaired, two-tailed Student’s t-test (B,D–F,K) or one-way ANOVA with Sidak’s multiple comparisons test (H,I).

Article Snippet: The primary antibodies used were specific for Flag (F1804, Sigma-Aldrich; 1:2000), Tcf7l1 (bs-12891, Bioss; 1:1000), HA (3724, Cell Signaling Technology; 1:1000), β-actin (66009-1-Ig, Proteintech; 1:1000), β-tubulin (66240-1-Ig, Proteintech; 1:1000), Oct4 (SC-5279, Santa Cruz; 1:1000), Sox2 (66411-1-Ig, Proteintech; 1:1000), Klf4 (381633, ZENBIO; 1:1000), Tbl1 (823294, ZENBIO; 1:1000), Tblr1 (R383062, ZENBIO; 1:1000), non-phospho (Active) β-catenin (Ser33/37/Thr41) (8814, Cell Signaling Technology; 1:1000), β-catenin (SC-7199, Santa Cruz; 1:1000) and ubiquitin (SC-8017, Santa Cruz; 1:1000).

Techniques: Over Expression, Western Blot, FLAG-tag, Plasmid Preparation, Control, Software, Quantitative RT-PCR, Staining, Expressing, Cell Culture, Transfection, Immunofluorescence, Two Tailed Test

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 1. Common commercially available ADNP antibodies give rise to non-specific binding. HEK293T, HeLa, SHSY-5Y and a lymphoblastoid control cell line (LCL) were lysed in RIPA buffer and used as protein samples for the assessment of the published ADNP antibodies. Samples were blocked and incubated in 5% blocking-grade non-fat dry milk/TBST with the optimized dilution listed in Table 3. The predicted molecular weight of ADNP is 124 kDa. However, only non-specific signals were detectable. GAPDH was used as a loading control. The datasheet of the tested antibodies indicated that whole or nuclear extracts from HeLa cells should be used as a positive control, which fails to raise a reliable ADNP signal in all tested antibody conditions.

Article Snippet: Here, the C-terminal ADNP antibody of Protein Technology detected multiple bands ranging from 124 to 250 kDa together with a possible proteolytic cleavage band of 50 kDa (Fig. 5A).

Techniques: Binding Assay, Control, Incubation, Blocking Assay, Molecular Weight, Positive Control

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 2. Verification of the specificity of an N-terminal ADNP antibody (Aviva Systems) by performing a blocking peptide competition assay. (A) HEK293T, HeLa, SHSY-5Y and a control lymphoblastoid cell line (LCL) were lysed in RIPA buffer and used as protein samples for the assessment of N-terminal antibody of Aviva systems in a 1:1000 dilution. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. The antibody recognizes ADNP specifically at 150 kDa in HEK293T, HeLa and SHSY-5Y cell lines, but a faint signal ranging from 75 to 150 kDa in the control LCL. (B) Western blot analysis of the blocking peptide competition assay. Supplementation of the immunization peptide in a 5 × excess to antibody concentration reduced the signal detected at 75- 150 kDa in all tested cell lines. Non-specific binding was detected after use of the immunization peptide presenting as a faint signal below the 37 kDa marker.

Article Snippet: Here, the C-terminal ADNP antibody of Protein Technology detected multiple bands ranging from 124 to 250 kDa together with a possible proteolytic cleavage band of 50 kDa (Fig. 5A).

Techniques: Blocking Assay, Competitive Binding Assay, Control, Molecular Weight, Western Blot, Concentration Assay, Binding Assay, Marker

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 3. A polyclonal N-terminal ADNP antibody from Aviva Systems detects ADNP specifically in murine and rat tissues and suggests proteolytic processing of the protein in the human brain. Cerebellum, frontal cortex or lobe, hippocampus and whole brains of control mice, rats and humans were lysed in RIPA buffer and used as protein samples for the assessment of N-terminal antibody of Aviva systems. (A–C) The predicted molecular weight of ADNP is 124 kDa. The antibody recognizes ADNP in a range of 145 kDa with (E) additional lower mass signal of 85 kDa in all human brain regions. (B–D–F) Western blot analysis of the blocking peptide competition assay. Supplementation of the immunization peptide in a 5 × excess to antibody concentration reduced the signal observed at 145 kDa in all tested cell lines. Importantly, the 85 kDa band suggestive for proteolytic cleavage as well as degraded ADNP signal disappeared completely after immunization peptide supplementation. GAPDH was used as a loading control.

Article Snippet: Here, the C-terminal ADNP antibody of Protein Technology detected multiple bands ranging from 124 to 250 kDa together with a possible proteolytic cleavage band of 50 kDa (Fig. 5A).

Techniques: Control, Molecular Weight, Western Blot, Blocking Assay, Competitive Binding Assay, Concentration Assay

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 4. Three independent commercially available C-terminal polyclonal ADNP antibodies detect ADNP specifically in different in vitro sample materials and show clear instability of the protein. HEK293T, HeLa, SHSY-5Y and a lymphoblastoid cell line (LCL) were lysed in RIPA buffer and used as protein samples for three different C-terminal ADNP antibodies. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. All the tested antibodies recognized ADNP with a molecular weight of 150 kDa. Samples were blocked and incubated in 5% blocking-grade non-fat dry milk/TBST with the optimized dilution listed in Table 3.

Article Snippet: Here, the C-terminal ADNP antibody of Protein Technology detected multiple bands ranging from 124 to 250 kDa together with a possible proteolytic cleavage band of 50 kDa (Fig. 5A).

Techniques: In Vitro, Control, Molecular Weight, Incubation, Blocking Assay

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 5. Different C-terminal ADNP antibodies detect ADNP in the range of 150 kDa and suggest proteolytic processing of the protein in the brain. Cerebellum, frontal cortex or lobe, hippocampus and whole brains of control mice, rats, and humans were lysed in RIPA buffer and used as protein samples for the assessment with three C-terminal antibodies with the optimized dilutions listed in Table 3. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. (A)C) Murine samples indicate detection of ADNP in the range of 150 kDa with bands suggesting proteolytic processing at 50 kDa. (D–F) Rat samples indicate detection of ADNP in the range of 150 kDa with bands indicating proteolytic processing at 82 kDa after incubation with the C-terminal Abcam antibody. (G–I) Human brain samples indicate detection of ADNP at different molecular weights of 124 – 150 kDa in the adult frontal lobe and hippocampus and highlight the antibody differences in detection of ADNP. The three tested antibodies showed strong band signals at lower molecular weights, which could indicate proteolytic cleavage or degradation of the protein.

Article Snippet: Here, the C-terminal ADNP antibody of Protein Technology detected multiple bands ranging from 124 to 250 kDa together with a possible proteolytic cleavage band of 50 kDa (Fig. 5A).

Techniques: Control, Molecular Weight, Incubation

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 6. Unambiguous detection of ADNP using homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines. mESCs containing either wild-type, homozygous mutants, or complete Adnp knockout were lysed in RIPA buffer and used as protein samples for the assessment with an N-terminal ADNP, 3x-DYKDDDDK, and C-terminal ADNP antibodies with the optimized dilutions listed in Table 1. GAPDH was used as a loading control. The predicted molecular weight of ADNP is 124 kDa. (A) The N-terminal antibody (Aviva Systems) recognizes ADNP in a range above its observed 150 kDa molecular weight with additional lower mass signal of 37—65 kDa in Adnp homozygous and parental control mESCs. (B) Supplementation of the immunization peptide in a 5 × excess to antibody concentration reduced all signals observed mESC lines, indicating that the N-terminal antibody does not bind ADNP specifically in mESCs. (C) Detection of wild- type and homozygous Adnp mutants by means of a C-terminal 3x-DYKDDDDK (Flag) epitope tag. Wild-type ADNP was detected in at 150 kDa in the C-terminal 3x-DYKDDDDK CRISPR/Cas9 engineered mESC line using a DYKDDDDK antibody. Truncated ADNP mutants, p.Tyr718* and p.Lys407Valfs*31, were detected at a lower molecular weight of 80 kDa, respectively 48 kDa. (D–F) Wild-type ADNP detection by means of three different C-terminal antibodies in mESC lines. Wild-type ADNP was detected with a strong signal at 150 kDa in the parental control line with a rather decreased signal in the C-terminal 3x-DYKDDDDK CRISPR/Cas9 engineered mESC line. Disappearance of the 150 kDa band was observed in the mESC line with complete Adnp homozygosity, indicating a reliable molecular weight of 150 kDa for ADNP.

Article Snippet: Here, the C-terminal ADNP antibody of Protein Technology detected multiple bands ranging from 124 to 250 kDa together with a possible proteolytic cleavage band of 50 kDa (Fig. 5A).

Techniques: CRISPR, Knock-Out, Control, Molecular Weight, Concentration Assay, FLAG-tag

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 7. Unambiguous detection of ADNP using an N-terminal GFPSpark and N-DYKDDDDK (Flag) tag expression vector. (A) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP- GFPSpark and mutated constructs using an anti-GFP antibody. (B) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP-GFPSpark and mutated constructs using the N-terminal ADNP antibody (Aviva Systems). (C) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP- DYKDDDDK (Flag) and mutated constructs using an anti-DYKDDDDK antibody. (D) Western blot analysis of HEK293T cell lysates overexpressing wild-type ADNP-DYKDDDDK and mutant constructs using the N-terminal ADNP antibody (Aviva Systems). The observed molecular weight of wild-type ADNP-GFPSpark is 175 kDa (including 25 kDa GFPSpark tag), respectively ADNP-DYKDDDDK 150 kDa, with each of their mutants showing a lower molecular weight as a consequence of the truncating mutations. Detection with antibodies for GFP, DYKDDDDK (Flag), and ADNP gave comparable results. GAPDH was used as a loading control in all experiments.

Article Snippet: Here, the C-terminal ADNP antibody of Protein Technology detected multiple bands ranging from 124 to 250 kDa together with a possible proteolytic cleavage band of 50 kDa (Fig. 5A).

Techniques: FLAG-tag, Expressing, Plasmid Preparation, Western Blot, Construct, Mutagenesis, Molecular Weight, Control

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 8. Western blotting of ADNP in a HCT116 colon cancer cell line, carrying the prevalent heterozygous p.Tyr719* mutation. HCT116 cells containing a wild-type and p.Tyr719* mutant allele were lysed in RIPA buffer and used as protein samples for the assessment with an N-terminal antibody, 3x-DYKDDDDK, HA-tag, and C-terminal ADNP antibodies with the optimized dilutions listed in Table 1. GAPDH was used as a loading control in all experiment. The predicted molecular weight of ADNP is 124 kDa. (A) The N-terminal antibody (Aviva Systems) recognizes ADNP in a range above its observed 150 kDa molecular weight an additional signal of 45 kDa, indicating proteolytic cleavage or non-specific binding. (B) Administration of the immunization peptide in a 5 × excess to antibody concentration reduced all signals, indicating that the N-terminal antibody does not bind ADNP specifically in HCT116 cells. (C) Detection of wild-type ADNP by means of the 3x-DYKDDDDK (Flag) epitope tag. Wild-type ADNP was detected in at 182 kDa in the 3xFlag-V5-loxP- neonGreen/3xHA-loxP-mCherry engineered line using a DYKDDDDK antibody, 32 kDa by tag insertion. (D) Detection of mutant ADNP by means of the HA-epitope tag. A truncated mutant p.Tyr719 ADNP protein was detected in at 105 kDa in the 3xFlag-V5-loxP-neonGreen/3xHA-loxP-mCherry engineered line using a HA-antibody, 25 kDa above its predicted molecular weight by tag insertion. Instability of the truncated protein was observed by a degrading smear. (E–G) Wild-type ADNP detection by means of three different C-terminal antibodies. Non-processed ADNP was detected with a strong signal at 150 kDa in the control line and at a molecular weight of 182 kDa in the genome-edited cell line. In both cases, a degrading smear was observed, indicating instability of the wild-type protein.

Article Snippet: Here, the C-terminal ADNP antibody of Protein Technology detected multiple bands ranging from 124 to 250 kDa together with a possible proteolytic cleavage band of 50 kDa (Fig. 5A).

Techniques: Western Blot, Mutagenesis, Control, Molecular Weight, Binding Assay, Concentration Assay, FLAG-tag

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 9. Western blotting of ADNP in human induced pluripotent stem cells (hiPSCs), carrying distinct heterozygous ADNP mutations mediated by CRIPSR/Cas9. (A, B) hiPSCs were lysed in RIPA buffer and analyzed by western blotting with the N-terminal antibody (Aviva Systems) with application of our blocking peptide competition assay. Here, no reliable ADNP signal was detected. The molecular weight of the ADNP mutant lines is expected to decrease to 127 kDa for the Asn832Lysfs*81, respectively to 48 kDa for the lys408Valfs*31 line. However, no signal is observed at the predicted weight for the mutations. (C–E) The C-terminal antibodies of Protein Technology, Abcam, and the Sarma Laboratory were able to visualize wild- type ADNP at 150 kDa. Possessing the desired epitope for mutant ADNP detection, the C-terminal antibody of Protein technology was not able to capture the predicted truncated protein. GAPDH was used as a loading control. (F) The ADNP signal was quantified determining the ratio of the wild-type protein in mutant to control cell lines. Here, the relative ADNP expression decreased in the Asn832Lysfs*81 cell line compared to the control, whereas mutant-to-wild-type expression ratio showed a higher signal with the antibodies of Protein Technology and Abcam in the lys408Valfs*31 cell line.

Article Snippet: Here, the C-terminal ADNP antibody of Protein Technology detected multiple bands ranging from 124 to 250 kDa together with a possible proteolytic cleavage band of 50 kDa (Fig. 5A).

Techniques: Western Blot, Blocking Assay, Competitive Binding Assay, Molecular Weight, Mutagenesis, Control, Expressing

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 10. Absence of a mutant ADNP protein after immunoblotting of different lymphoblastoid cell lines from Helsmoortel-Van der Aa syndrome patients. (A) LCLs of four control subjects and six patients were lysed in RIPA buffer and analyzed by western blotting with the N-terminal antibody (Aviva Systems). The expected wild-type ADNP signal presented at 150 kDa together with two non-specific bands at 50 kDa and 75 kDa with no difference in expression (p = 0.42; ns) of the wild-type protein. However, the ADNP mutants at a lower molecular weight of 127 kDa for the Asn832Lysfs*81 and Leu831Ilefs*82 mutations, respectively to 45 kDa for the Ser404* mutation, and to 10 kDa for the cell line carrying the Gln40* mutation could not be visualized. (B) Administration of the immunization peptide in a 5 × excess to antibody concentration reduced all signals, indicating that the N-terminal antibody recognized ADNP specifically in LCLs alongside non-specific band signals. (C-E) C-terminal antibodies detected wild-type ADNP at a molecular weight of 150 kDa. No mutant ADNP was observed with the antibody of Protein Technology which is capable to recognize a part of the truncated Asn832Lysfs*81 and Leu831Ilefs*82 mutations. (F) All C-terminal antibodies visualized wild-type ADNP at 150 kDa, with only the Abcam (p = 0.04; *) and Sarma Laboratory (p = 0.02; *) antibodies showing the expected reduction of ADNP in LCLs of Helsmoortel-Van der Aa syndrome patients. GAPDH was used as a loading control.

Article Snippet: Here, the C-terminal ADNP antibody of Protein Technology detected multiple bands ranging from 124 to 250 kDa together with a possible proteolytic cleavage band of 50 kDa (Fig. 5A).

Techniques: Mutagenesis, Western Blot, Control, Expressing, Molecular Weight, Concentration Assay

Journal: Scientific reports

Article Title: Tracing the invisible mutant ADNP protein in Helsmoortel-Van der Aa syndrome patients.

doi: 10.1038/s41598-024-65608-x

Figure Lengend Snippet: Figure 11. Wild-type and mutant ADNP enrichment through immunoprecipitation. The N-terminal sc-F5 ADNP IP-competent antibody was crosslinked to agarose beads and sequentially eluted in fractions (input; flow-through; three consecutive washes, W1-W3; and the immunoprecipitated fracted. IgG non-reactive beads were used as a negative control. In each lane, 20 μg of protein was separated by SDS-PAGE electrophoresis. GAPDH has been used as loading control for all western blots, and critical assessment of the accuracy of the IP method. (A) Immunoprecipitation assay of recombinant wild-type (WT) ADNP and truncating mutants (p.Tyr719*; p.Arg730*; p.Asn832Lysfs*81) in HEK293T overexpression lysates. (B) Immunoprecipitation assay of native wild-type (WT) ADNP and truncating mutants in protein extracts of LCLs derived from a control subject (CTR) and patients with the p.Ser404*, p.Leu831Ilefs*82, or p.Asn832Lysfs*81 ADNP mutation.

Article Snippet: Here, the C-terminal ADNP antibody of Protein Technology detected multiple bands ranging from 124 to 250 kDa together with a possible proteolytic cleavage band of 50 kDa (Fig. 5A).

Techniques: Mutagenesis, Immunoprecipitation, Negative Control, SDS Page, Electrophoresis, Control, Western Blot, Recombinant, Over Expression, Derivative Assay

Journal: Redox biology

Article Title: Protein S-glutathionylation confers cellular resistance to ferroptosis induced by glutathione depletion.

doi: 10.1016/j.redox.2025.103660

Figure Lengend Snippet: Fig. 3. Decreased glutathione pools by CHAC1 reduces protein-SSG and aggravates APAP-induced hepatotoxicity and ferroptosis in APAP-injured mice liver. (A) Quantification of GSH in the liver tissues of Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP, and Chac1−/−Ad-CHAC1 mice treated with saline or 300 mg/kg APAP for 2 and 6 h (Data are mean ± SEM of n = 3 and 5 mice/group, t-test). (B) Quantification of GSSG in the liver tissues of Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP, and Chac1−/−Ad- CHAC1 mice treated with saline or 300 mg/kg APAP for 2 and 6 h (Data are mean ± SEM of n = 3 and 5 mice/group, t-test). (C) Western blot analysis of S-glu tathionylated proteins in the liver tissues of Chac1+/+ Ad-GFP and Chac1−/−Ad-GFP mice treated with 300 mg/kg APAP for 2 and 6 h. GAPDH was used as an internal reference, followed by quantification of relative levels of S-glutathionylated proteins after 6 h of APAP treatment (Data are mean ± SEM of n = 3 mice/ group, t-test). (D) Western blot analysis of S-glutathionylated proteins and CHAC1-FLAG protein in the liver tissues of Chac1−/−Ad-GFP and Chac1−/−Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h. GAPDH was used as an internal reference. Statistical chart shows the relative expression levels of S-glutathionylated proteins 6 h after APAP treatment (Data are mean ± SEM of n = 4 mice/group, t-test). (E) Serum levels of ALT and AST in Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP, and Chac1−/−Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h (Data are mean ± SEM of n = 5 mice/group, t-test). (F) H&E staining, 4-HNE protein adduct staining in liver tissues of Chac1+/+ Ad-GFP, Chac1−/−Ad-GFP and Chac1−/−Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h. Scale bars = 200 μm. The area of liver injury and immunohistochemical score for 4-HNE protein adduct staining were quantified (Data are mean ± SEM of n = 5 mice/group, t-test). APAP, acetaminophen; GSH, glutathione; GSSG, oxidized glutathione; ALT, alanine aminotransferase; AST, aspartate aminotransferase; H&E, haematoxylin and eosin; Chac1−/−, Chac1- deficient; Chac1+/+, wild-type controls.

Article Snippet: For Western blot, lysates were probed with specific antibodies against CHAC1 (Proteintech, Cat. # 15207-1-AP, 1:1000), glutathione (Virogen, Cat. # 101-A, 1:1000), ARF6 (Affinity, Cat. # DF6170, 1:1000), TFRC (Abcam, Cat. # ab269513, 1:2000), 4-hydroxynonenal (4-HNE, Abcam, Cat. # ab46545), FLAG-tag (Sigma-Aldrich, Cat. #F1804, 1:3000), and Myctag (Santa Cruz Biotechnology, Cat. # sc-40, 1:1000). β-actin (Proteintech, Cat. # 66009-1-Ig, 1:10000), and GAPDH (Proteintech, Cat. # 10494-1-AP, 1:5000) were used as loading control.

Techniques: Saline, Western Blot, Expressing, Staining, Immunohistochemical staining

Journal: Redox biology

Article Title: Protein S-glutathionylation confers cellular resistance to ferroptosis induced by glutathione depletion.

doi: 10.1016/j.redox.2025.103660

Figure Lengend Snippet: Fig. 4. Redox proteomic analysis reveals that the S-glutathionylation of Cys90 on ARF6 is regulated by CHAC1 in ferroptotic PMHs induced by APAP. (A) Flowchart outlining the key experimental procedures for proteomic analysis of S-glutathionylation. (B) Scatter plot illustrating the distribution of differential modification sites, sorted by the ratio of Ad-GFP + APAP/Ad-GFP. Red dots indicating up-regulation of significant differences, blue dots indicating down-regulation of significant differences, and grey dots indicating no significant differences (CV < 0.1, fold change ≥1.2). (C) Scatter plot showing the distribution of differential modification sites, sorted by the ratio of Ad-CHAC1 + APAP/Ad-GFP + APAP. Red dots indicating up-regulation of significant differences, blue dots indicating down-regulation of significant differences and grey dots indicating no significant differences (CV < 0.1, fold change ≥1.2). (D) Venn diagram showing differentially modified sites under both APAP stimulation and CHAC1 overexpression (Fold change ≥1.2). (E) The heat map illustrating the union of differential modification sites in Ad-GFP, Ad-GFP + APAP, Ad-CHAC1, and Ad-CHAC1 + APAP comparison groups (CV < 0.1, fold change ≥1.2). (F) Scatter plot showing differentially modified sites under both APAP stimulation and CHAC1 overexpression; the order was sorted by the ratio of Ad-GFP + APAP/Ad-GFP (CV < 0.1, fold change ≥1.2). (G) Two-stage mass spectrometry of the glutathionylated peptide from ARF6 in PMHs. The secondary mass spectrum shows fragment ion information of the ARF6 C90 peptide segment. (H) Histogram showing the relative modification abundance of ARF6 C90 in different treatment groups, with glutathionylated peptides identified and quantified by LC-MS/MS (All values were normalized by the mean of the AdGFP-CON group, data are mean ± SEM of n = 2 biologically independent samples). (I) IP assay showing the expression of S-glutathionylated ARF6 in 293T cells overexpressing Myc-tagged ARF6. Whole cell lysates were used to confirm the expression of ARF6. (J) Two-stage mass spectrometry of the glutathionylated peptide from ARF6 in 293T cells overexpressing Myc-tagged ARF6. The secondary mass spectrum shows fragment ion infor mation of the ARF6 C90 peptide segment. PMH, primary mouse hepatocyte; IP, immunoprecipitation.

Article Snippet: For Western blot, lysates were probed with specific antibodies against CHAC1 (Proteintech, Cat. # 15207-1-AP, 1:1000), glutathione (Virogen, Cat. # 101-A, 1:1000), ARF6 (Affinity, Cat. # DF6170, 1:1000), TFRC (Abcam, Cat. # ab269513, 1:2000), 4-hydroxynonenal (4-HNE, Abcam, Cat. # ab46545), FLAG-tag (Sigma-Aldrich, Cat. #F1804, 1:3000), and Myctag (Santa Cruz Biotechnology, Cat. # sc-40, 1:1000). β-actin (Proteintech, Cat. # 66009-1-Ig, 1:10000), and GAPDH (Proteintech, Cat. # 10494-1-AP, 1:5000) were used as loading control.

Techniques: Modification, Over Expression, Comparison, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Expressing, Immunoprecipitation

Journal: Cell biology and toxicology

Article Title: Targeting p38γ synergistically enhances sorafenib-induced cytotoxicity in hepatocellular carcinoma.

doi: 10.1007/s10565-024-09979-x

Figure Lengend Snippet: Fig. 1 P38γ/MAPK12 is a targeted driving fac- tor for the development of hepatocellular carci- noma. The expression level of p38γ in tumor tissues is related to the prognosis of patients and p38γ washighly expressed in hepatocellular carcinoma cell lines. The data of p38α (A, B), p38β (C, D), p38γ (E, F) and p38δ (G, H) in the Ualcan database website were analyzed. I, J: Western Blot was used to detect the expression of p38γ in nor- mal liver cell line AML-12 and liver cancer cell lines Huh-7, PLC/PRF/5, Hep-G2 and BEL-7404. [UALCAN (http://ualcan. path.uab.EdU) is an online analysis tool in the TCGA database. We used this database to analyze the expression of MAPK family (p38α, p38β, p38γ, p38δ) in hepatocellular carci- noma and its correlation with patient prognosis]

Article Snippet: Antibodie: p38 gamma/MAPK12 (Abcam, ab205926), GAPDH (Zen Bioscience, 200,306), LC3B (Abcam, ab192890), p62 (Abcam, ab109012), Phospho-p38 MAPK (Thr180/Tyr182) (CST, 9211), FLAG tag (Prospec, ANT-146), ATG5 (Proteintech, 10181–2-AP).

Techniques: Expressing, Western Blot