Journal: Development (Cambridge, England)

Article Title: IRX3/5 regulate mitotic chromatid segregation and limb bud shape

doi: 10.1242/dev.180042

Figure Lengend Snippet: IRX3 associates with SMC1 and CUX1 in vivo. (A) Lysate from 30 WT E10.5 hindlimb buds was immunoprecipitated (IP) with anti-IRX3 and immunoblotted against SMC1, SMC3 or CUX1. Three separate co-IP experiments were performed using multiple limb buds on each occasion. Representative full-length western blots (WB) are shown, and arrows indicate expected band size. (B) IRX3 protein localised in anterior-proximal (AP), but not posterior-distal (PD), nuclei in mesoderm of 29 som. limb bud. (C) STED microscopy revealed that IRX3 puncta contacted those of SMC1 (anterior versus posterior, P=0.008) and CUX1 (anterior versus posterior, P=0.005) in anterior mesoderm of the hindlimb field at 29-32 som. (25-30 cells, n=3 embryos, unpaired, two-tailed t-test, error bars indicate s.e.m.). (D,E) PLA in vivo demonstrated physical association of IRX3 with SMC1 (8/82, 9.7%; D) and CUX1 (8/64, 12.5%; E) in anterior half mesoderm at 29-30 som. (n=3 embryos per condition). Scale bars: 400 µm (B, left); 20 µm (B, right); 5 µm (C-E).

Article Snippet: Antibodies company and catalogue number IRX3 [Novus Biologicals, 1D7, H0079191-M05, mouse, 1:250 – used for immunofluorescence (IF)]; IRX3 (Santa Cruz Biotechnology, G-6, 166877, mouse, 1:100 – used for co-IP in vivo ); SMC1 (Abcam, ab21583, rabbit, 1:250 IF, 1:1000 – used for immunoblotting (IB); SMC3 (Novus Biological, NB100-207 IF, in vitro IB; Abcam, ab9263, 1:1000 in vivo IB); NIPBL (Santa Cruz Biotechnology, C-9, sc-374625, 1:1000); CUX1 (Abcam, ab140042, mouse, 1:250 IF, in vitro IB; Abcam, ab230844, rabbit, 1:2500 in vivo IB); IgG (NA931V, GE Healthcare, 1:100) or donkey anti-rabbit IgG (NA934V, GE Healthcare, 1:100) co-IP in vitro , IgG (Santa Cruz Biotechnology, 2343, 1:100) co-IP in vivo ; Caspase-3 (BD Biosciences, 559565, 1:250); F-Actin (Alexa Fluor 633 Phalloidin, Thermo Fisher Scientific, A22284, 1:1000).

Techniques: In Vivo, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Microscopy, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: IRX3/5 regulate mitotic chromatid segregation and limb bud shape

doi: 10.1242/dev.180042

Figure Lengend Snippet: IRX3/5 maintain cohesin subunits and CUX1 in vivo. (A,B) Immunofluorescence intensities of SMC1, NIPBL, SMC3 and CUX1 were diminished in the absence of Irx3/5 in anterior (A), but not posterior (B), limb bud mesoderm. (C) Quantification of A and B (29-32 som.; n=3 embryos per condition; *P=0.008 for anterior SMC1, *P=0.003 for anterior NIPBL, *P=0.004 for anterior SMC3, *P=0.004 for anterior CUX1; unpaired, two-tailed t-test, error bars indicate s.e.m.). (D,E) Transcription of Smc1a, Smc1b and Cux1 were unchanged by real time RT-PCR in anterior and posterior limb bud tissue in Irx3/5+/− and Irx3/5−/− embryos (29-30 som., n=3 embryos for each of three independent experiments). Error bars indicate s.e.m. Scale bars: 20 µm.

Article Snippet: Antibodies company and catalogue number IRX3 [Novus Biologicals, 1D7, H0079191-M05, mouse, 1:250 – used for immunofluorescence (IF)]; IRX3 (Santa Cruz Biotechnology, G-6, 166877, mouse, 1:100 – used for co-IP in vivo ); SMC1 (Abcam, ab21583, rabbit, 1:250 IF, 1:1000 – used for immunoblotting (IB); SMC3 (Novus Biological, NB100-207 IF, in vitro IB; Abcam, ab9263, 1:1000 in vivo IB); NIPBL (Santa Cruz Biotechnology, C-9, sc-374625, 1:1000); CUX1 (Abcam, ab140042, mouse, 1:250 IF, in vitro IB; Abcam, ab230844, rabbit, 1:2500 in vivo IB); IgG (NA931V, GE Healthcare, 1:100) or donkey anti-rabbit IgG (NA934V, GE Healthcare, 1:100) co-IP in vitro , IgG (Santa Cruz Biotechnology, 2343, 1:100) co-IP in vivo ; Caspase-3 (BD Biosciences, 559565, 1:250); F-Actin (Alexa Fluor 633 Phalloidin, Thermo Fisher Scientific, A22284, 1:1000).

Techniques: In Vivo, Immunofluorescence, Two Tailed Test, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: a Demonstration of the PTMs locus of IGF2BP2 by the PhosphoSitePlus database. b Flowchart showing the route to construct the siRNA screen; IGF2BP2 expression was assessed by Western blot upon DUB knockdown, with densitometric analysis of band gray values. c A positive correlation was observed between IGF2BP2 and USP9X protein levels in Pan-Cancer Proteome Atlas (TPCPA)-BRCA TNBC ( R = 0.28, P = 0.031), with missing values imputed using a k-nearest neighbors- based algorithm. d IGF2BP2 activity is higher in USP9X-high tumors than in USP9X-low tumors in the FUSCC TNBC cohort. e , f Expression levels of USP9X in MDA-MB-231 and Hs 578T cells transfected with knockdown USP9X (shUSP9X) and blank control (shRNA-NC) were detected by qRT-PCR and Western blot. g , h Western blot analysis of IGF2BP2 protein levels in MDA-MB-231 and Hs 578T cells stably transfected with shUSP9X or control shRNA (shRNA-NC) following treatment with 50 μg/ml cycloheximide (CHX) for the indicated time points (0, 2, 4, 6, 8 h). Quantification of grayscale values from all experimental groups was performed to determine the protein degradation half-life (t₁/₂) of IGF2BP2. i , j HEK 293T cells were co-transfected with HIS-IGF2BP2 and FLAG-USP9X, followed by treatment with 50 μg/ml CHX to inhibit protein synthesis. Cells were harvested at the indicated time points (0, 2, 4, 6, 8 h), and protein levels were analyzed by Western blot using anti-HIS (for IGF2BP2) and anti-FLAG (for USP9X) antibodies. Quantification of grayscale values from all experimental groups was performed to determine the protein degradation half-life (t₁/₂) of HIS-IGF2BP2. Error bars indicate mean ( n = 3) ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: Construct, Expressing, Western Blot, Knockdown, Activity Assay, Transfection, Control, shRNA, Quantitative RT-PCR, Stable Transfection, Standard Deviation

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: a Cell lysates of MDA-MB-231 and Hs 578T were pulled down by IP using antibodies against USP9X and IGF2BP2, followed by Western blot detection. b , c Confocal images showing co-localization of USP9X (red) and IGF2BP2 (green) in MDA-MB-231 and Hs 578T cells, and nuclei were restained with DAPI (blue), followed by quantitative analysis of fluorescence intensity. d Domain organization schematics of FLAG-USP9X, HIS-IGF2BP2 and their engineered truncation constructs, depicting functional domains and truncation boundaries. e HEK 293T cells were co-transfected with HIS-IGF2BP2 and FLAG-tagged USP9X full-length or truncated mutants. Cell lysates were analyzed by IP with FLAG beads and then detected by Western blot with antibodies against HIS and FLAG. f HEK 293T cells were cotransfected with FLAG-USP9X and HIS-tagged full-length or truncated mutants of IGF2BP2. Cell lysates were analyzed by IP with HIS beads and then detected by Western blot with antibodies against FLAG and HIS. g HDock ( http://hdock.phys.hust.edu.cn/ ) analysis showing details of the interaction between USP9X-M3 and IGF2BP2-M3, the protein models were retrieved and downloaded at UniProt ( https://www.uniprot.org/ ). h Purified GST-USP9X-M3 and HIS-IGF2BP2 were incubated in binding buffer at 4 °C for 2 h, Complexes were pulled down with HIS/GST magnetic beads, Input (10%) and bound fractions were analyzed by Western blot.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: Western Blot, Fluorescence, Construct, Functional Assay, Transfection, Purification, Incubation, Binding Assay, Magnetic Beads

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: a , b Total proteins were extracted from control and USP9X-knockdown MDA-MB-231 and Hs 578T cells treated with DMSO, MG132, or CQ for 8 h. IGF2BP2 protein levels were analyzed by Western blot, and band intensities were quantified for statistical comparison. Error bars indicate mean ( n = 3) ± standard deviation. c , d MDA-MB-231 and Hs 578T cells were co-transfected with HA-Ub along with either control siRNA, USP9X-targeting siRNA (siUSP9X), or FLAG-USP9X plasmid. Following MG-132 (20 μM, 6 h) treatment to stabilize ubiquitinated proteins, IGF2BP2 was immunoprecipitated and its ubiquitination status was analyzed by Western blot using anti-HA antibody. e , f HEK 293T cells were cotransfected using HIS-tagged IGF2BP2 with HA-Ub, Lys48 only, Lys63 only, Lys48R, Lys63R plasmids, respectively, and after treatment with 20 μM MG-132 for 6 h. HIS pull-down assays were performed and ubiquitination patterns were analyzed by immunoblotting with anti-HA for ubiquitin conjugates and anti-HIS for IGF2BP2 loading control. g HA-Ub wild-type or Ub-Lys48R was transfected in MDA-MB-231 and Hs 578T cells in control or knockdown USP9X groups. After 72 h, total protein was extracted and IGF2BP2 levels were analyzed by Western blot using anti-IGF2BP2 antibody, with USP9X detection serving as knockdown verification. h HEK293T cells were co-transfected with FLAG-USP9X, HA-Ub, and either wild-type HIS-IGF2BP2 or its lysine-to-arginine (K-R) mutants. Following MG-132 treatment (20 μM, 6 h) to stabilize ubiquitinated proteins, HIS-tagged IGF2BP2 was immunoprecipitated and ubiquitination patterns were analyzed by immunoblotting with anti-HA (ubiquitin), anti-HIS (IGF2BP2), and anti-FLAG (USP9X). i HEK 293T cells were cultured for 72 h after co-transfection with empty or siUSP9X using wild-type Ub, HIS-IGF2BP2 or HIS-IGF2BP2487/583R plasmids, and total proteins were collected and subjected to Western blot. Quantification of HIS-IGF2BP2 protein levels normalized to β-Actin. j HIS-tagged IGF2BP2-WT or HIS-IGF2BP2-487/583R was used to co-transfect with HA-Ub and FLAG-USP9X, and ubiquitin ligation of IGF2BP2 was analyzed after collection of total proteins after treatment with 20 μM MG-132 for 6 h and IP pull-down using HIS antibody.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: Control, Knockdown, Western Blot, Comparison, Standard Deviation, Transfection, Plasmid Preparation, Immunoprecipitation, Ubiquitin Proteomics, Cell Culture, Cotransfection, Ligation

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: a Cell lysates of MDA-MB-231 and Hs 578T were pulled down by IP using antibodies against WWP1 and IGF2BP2, followed by Western blot detection. b HEK293T cells were co-transfected with HIS-tagged IGF2BP2 and FLAG-tagged WWP1, treated with CHX (50 μg/ml) and total proteins were collected at the corresponding time and then detected by Western blot, and the gray values were calculated for statistical analysis. Error bars indicate mean ( n = 3) ± standard deviation. c – f MDA-MB-231 and Hs 578T were co-transfected with HA-Ub using the indicated controls, siWWP1 or FLAG-tagged WWP1, and after treatment with 20 μM MG-132 for 6 h. IGF2BP2 was immunoprecipitated and its ubiquitination status was analyzed by immunoblotting with anti-HA antibody (for ubiquitin conjugates) and anti-IGF2BP2 antibody (for protein input control). g HA-Ub wild-type or Ub-Lys48R was transfected in MDA-MB-231 and Hs 578T cells transfected with empty vector or FLAG-tagged WWP1 groups, and the total proteome was collected after 72 h and detected by Western blot using IGF2BP2 and FLAG antibodies. h HEK 293T cells were cultured with wild-type Ub, HIS-IGF2BP2 or HIS-IGF2BP2-487/583R plasmids co-transfected with null or FLAG-WWP1 for 72 h. Total proteins were collected and subjected to Western blot. i HEK293T cells were co-transfected with HIS-IGF2BP2 and empty vector (control), FLAG-WWP1, FLAG-USP9X, or both FLAG-WWP1 and FLAG-USP9X, along with wild-type ubiquitin. After 72 h, total protein was extracted and IGF2BP2 expression levels were analyzed by immunoblotting with anti-HIS antibody, while ectopic WWP1 and USP9X expression was verified using anti-FLAG antibody. j HEK 293T cells were cotransfected with HA-Ub, HIS-IGF2BP2 with empty or FLAG-USP9X or FLAG-USP9X and FLAGWWP1 and treated with 20 μM MG-132 for 6 h. After treatment with HIS antibody, the cells were subjected to IP pull-down and the ubiquitination levels of HIS-IGF2BP2 were ultimately assessed by Western blot analysis.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: Western Blot, Transfection, Standard Deviation, Immunoprecipitation, Ubiquitin Proteomics, Control, Plasmid Preparation, Cell Culture, Expressing

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: a CCK-8 assay was used to examine the IC 50 value of Cisplatin (CDDP, MCE, HY-17394). b Scatter plot of IGF2BP2 and USP9X expression in individual patients, with color indicating cisplatin response status. Dotted lines represent median values of IGF2BP2 and USP9X expression, used to define high and low expression subgroups. Fisher ’s exact test was used to compare the IGF2BP2High/USP9XHigh group against each of the other three subgroups. The response rate was significantly higher than that in the IGF2BP2Low/USP9XHigh group ( P = 0.003) and the IGF2BP2Low/USP9XLow group ( P = 0.020), and showed a non-significant trend toward higher response compared to the IGF2BP2High/USP9XLow group ( P = 0.179). Heatmap colors indicate response rates. c , d . Expression levels of USP9X, WWP1 and IGF2BP2 in MDA-MB-231 and Hs 578T cells from saline control (NC) and cisplatin drug-treated (CDDP) groups were detected by qRT-PCR and Western blot. e HA-Ub was transfected in NC and CDDP group cells, respectively, and IP pull-down was performed using IGF2BP2 antibody and detected by Western blot. f , g Confocal images showing co-localization of USP9X or WWP1 (red) and IGF2BP2 (green) in MDA-MB-231 and Hs 578T cells after 4 h treatment with saline or CDDP for 4 h, with nuclei were restained with DAPI (blue). h His-tagged IGF2BP2-M3 was overexpressed in IGF2BP2-knockdown MDA-MB-231 and Hs 578T cells treated with cisplatin or saline control. Total proteins were IP using anti-HIS antibody, followed by Western Blot assays with antibodies against USP9X, WWP1, and HIS-tag. i Biotin-labeled cisplatin probes were synthesized for pull-down assay and the enriched proteins were used for mass spectrometry identification. The identification results comprehensively listed all unique peptide sequences specific to USP9X. j Cell lysates from MDA-MB-231, Hs 578T, and HEK293T cells were incubated with biotin-labeled cisplatin probes or biotin-only controls coupled to streptavidin magnetic beads. After pull-down and denaturation, captured proteins were analyzed by Western blot with USP9X antibody to specifically detect cisplatin-USP9X interactions. k Four FLAG-tagged USP9X truncation mutants were purified as recombinant proteins for in vitro binding assays. These proteins were incubated with streptavidin magnetic beads conjugated to either biotin-cisplatin or biotin alone. Bound proteins were eluted and analyzed by immunoblotting with anti-FLAG antibody. Error bars indicate mean ( n = 3) ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: CCK-8 Assay, Expressing, Saline, Control, Quantitative RT-PCR, Western Blot, Transfection, Knockdown, Labeling, Synthesized, Pull Down Assay, Mass Spectrometry, Incubation, Magnetic Beads, Purification, Recombinant, In Vitro, Binding Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: a Working model of IGF2BP2 targeting MYC and CDK6 mRNAs in an m6A-dependent manner to promote TNBC progression. b – e qRT-PCR was performed to measure the transcript levels of CDK6 and MYC within IGF2BP2 or IgG immunocomplexes in saline or cisplatin-treated MDA-MB-231 and Hs 578T cell lysates. Relative levels of all genes were normalized with IgG. f After treatment with CHX (50 μg/ml), the corresponding total proteins were collected at different time points of cisplatin addition and protein abundance of IGF2BP2 and CDK6 was detected by Western blot. g Protein abundance of IGF2BP2 and CDK6 was made to be converted to gray values and analyzed statistically. h mRNA half-life (t1/2) was measured in cisplatin-treated vs control cells using actinomycin D (5 μg/mL) chase (0, 0.5, 1 h) followed by qRT-PCR. i MDA-MB-231 and Hs 578 T cells were treated with either saline or cisplatin for 4 h. IGF2BP2-containing complexes were immunoprecipitated from cell lysates and analyzed by immunoblotting for interacting partners (HUR and EIF4A1), with IGF2BP2 serving as the immunoprecipitation control. Error bars indicate mean ( n = 3) ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: Quantitative RT-PCR, Saline, Quantitative Proteomics, Western Blot, Control, Immunoprecipitation, Standard Deviation

Journal: Cell Death & Disease

Article Title: The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity

doi: 10.1038/s41419-025-08038-5

Figure Lengend Snippet: Mechanistically, USP9X and WWP1 coordinately control IGF2BP2 stability through ubiquitination modulation. Cisplatin (CDDP) binding to USP9X disrupts this balance. Therapeutically, combining WP1130 (USP9X inhibitor) with cisplatin ablates IGF2BP2’s m6A-binding activity toward MYC and CDK6 mRNAs, providing an effective strategy against TNBC progression.

Article Snippet: Further, we purified the HIS-tagged IGF2BP2 full length protein and performed in vitro protein interaction pull-down experiments with the purified protein of the GST-tagged USP9X M3 segment (HY-P701431, MCE, NJ, USA) to further clarify its interconnections (Fig. ).

Techniques: Control, Ubiquitin Proteomics, Binding Assay, Activity Assay

Journal: Nature Communications

Article Title: Specific inhibition of the Survivin–CRM1 interaction by peptide-modified molecular tweezers

doi: 10.1038/s41467-021-21753-9

Figure Lengend Snippet: a 293T cell lysate with overexpressed Survivin142-HA was preincubated with either unmodified tweezer (TW), TW-ELTL, TW-ELTLGEFL, or a scrambled peptide-modified tweezer, TW-LFEEGLLT, at concentrations ranging from 0.01 to 200 µM. GST-CRM1 was mixed with either non- or preincubated cell lysates in the presence of recombinant RanQ69L and dGTP to allow complex assembly. GST-CRM1 and interacting proteins were pulled by GSH-Sepharose beads. Proteins in input and beads samples were analyzed via immunoblotting with antibodies specific for GST or HA. For each tweezer, samples derive from the same experiment and gels/blots were processed in parallel. Direct comparison for this exact concentration range was performed once for TW, TW-ELTL, and TW-ELTLGEFL and for TW-LFEEGLLT in three technical replicates. b , c Atto488-labeled Survivin120 was preincubated with CRM1_1-1062VLV430AAA mutant in a ratio of 1:5 and titrated with supramolecular tweezers up to approx. 180 µM. Fluorescence anisotropy was measured ( n = 1) ( b ), and IC50 values were determined from the resulting curves ( c ). TW light blue/ triangles, TW-ELTL blue/squares, TW-ELTLGEFL dark blue/circles. Source data are provided as a Source Data file.

Article Snippet: Next, membranes were incubated in primary antibodies against HA (anti-HA, mouse monoclonal, BioLegend, Covance MMS-101R, 1:1,000), GST (anti-GST, mouse monoclonal, Santa Cruz Biotechnology Inc., sc-57753, 1:1000) or CRM1 (anti-CRM1, rabbit polyclonal, Novus Biologicals Ltd., NB100-79802, 1:10,000) overnight at 4 °C.

Techniques: Modification, Recombinant, Western Blot, Comparison, Concentration Assay, Labeling, Mutagenesis, Fluorescence

Journal: Nature Communications

Article Title: Specific inhibition of the Survivin–CRM1 interaction by peptide-modified molecular tweezers

doi: 10.1038/s41467-021-21753-9

Figure Lengend Snippet: Titration of 300 μM TW ( a ) in the cell with 321 µM Survivin120 K90/103T in the syringe. Titration of 100 µM TW-ELTL ( b ) and TW-ELTLGEFL ( c ) in the cell with 2.5 mM Survivin120 K90/103T in the syringe. All titrations were performed in PBS, pH 7.4 at 25 °C. Graphs represent one representative example each from three independent experiments ( n = 3). The black lines in the bottom panels are the best fit of the data to a one set of sites model. The heat of dilution was subtracted as constant. For thermodynamic data derived from the graphs see SI . FAM-labeled unmodified tweezer molecule ( d ), TW-ELTL ( e ), and TW-ELTLGEFL ( f ) (0.2 µM) were titrated with either Survivin120 (200 µM, circles) or Survivin120 K90/103T (400 µM, squares). Survivin120 K90/103T showed greatly reduced tweezer affinities (lower curves). d – f Data are presented as mean values ± SD with n = 3 independent experiments. g Pull-down results after immunostaining. GST-Survivin120-WT or GST-Survivin120-K90/103T were incubated with 50 µM respective tweezer molecule or ELTL/ ELTLGEFL peptides w/o tweezer. GST-Survivin120- or GST-Survivin120-K90/103T-loaded beads were mixed with CRM1 and RanQ69L prey proteins as well as dGTP to allow export complex assembly. Proteins in input and bead samples were analyzed via immunoblotting with antibodies specific for CRM1 or GST. WT, wildtype. One representative example of two independent biological replicates is shown. Samples derive from the same experiment and gels/blots were processed in parallel. h Quantification of two independent pull-down experiments. After subtraction of the CRM1 negative control from the pulled CRM1 intensity, the latter is normalized by the GST–Survivin intensity and afterwards normalized by the CRM1 intensity without tweezer incubation. Export complex assembly is only compromised by peptide tweezers in the wildtype Survivin120, but not in the mutant. No ligand: black; TW: light blue; TW-ELTL: blue; TW-ELTLGEFL: dark blue; ELTL peptide: light gray; ELTLGEFL peptide: dark gray. Source data are provided as a Source Data file.

Article Snippet: Next, membranes were incubated in primary antibodies against HA (anti-HA, mouse monoclonal, BioLegend, Covance MMS-101R, 1:1,000), GST (anti-GST, mouse monoclonal, Santa Cruz Biotechnology Inc., sc-57753, 1:1000) or CRM1 (anti-CRM1, rabbit polyclonal, Novus Biologicals Ltd., NB100-79802, 1:10,000) overnight at 4 °C.

Techniques: Titration, Derivative Assay, Labeling, Immunostaining, Incubation, Western Blot, Negative Control, Mutagenesis

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: USP25 Inhibits Neuroinflammatory Responses After Cerebral Ischemic Stroke by Deubiquitinating TAB2.

doi: 10.1002/advs.202301641

Figure Lengend Snippet: Figure 5. USP25 deficiency enhances LPS-induced cytokine production in microglia. A–C) Transcription of A) TNF-𝛼, B) IL-6, and C) IL-1𝛽in control and USP25−/−BV2 cells was analyzed by qRT-PCR after stimulation with LPS (500 ng ml−1) for the indicated time periods. D–F) Transcription of D) TNF-𝛼, E) IL-6, and F) IL-1𝛽in control and USP25−/−primary microglia was detected by qRT-PCR after stimulation with LPS (500 ng ml−1) for 3 h. G–I) BV2 cells were pretreated with AZ1 (5 μm) for 2 h, followed by stimulation with LPS (500 ng ml−1) for 3 h. The transcription of G) TNF-𝛼, H) IL-6, and I) IL-1𝛽 was detected by qRT-PCR. All data represent the mean ± SEM. n = 3 per group. * P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: For USP25 inhibition, cells were treated with 5 μm AZ1 (MedChemExpress, NJ, USA).

Techniques: Control, Quantitative RT-PCR