Journal: Glia

Article Title: Impairment of protein degradation and proteasome function in hereditary neuropathies

doi: 10.1002/glia.23251

Figure Lengend Snippet: (A) The DUBs USP5, USP14 and UCH37 are increased on the 26S proteasome in S63del L. Proteasomes were incubated with HA-Ub-VS to modify the active site cysteines of the associated DUBs that copurified with them. Levels of modification, and thus activity of the DUB, were analyzed by western blot for HA and normalized to both the 20S ɑ subunits and the 19S subunit Rpt1. 5 HA-positive bands were detected and re-probing the blots with DUB-specific antibodies enabled the identification of Band 2 as USP5, Band 3 as USP14, and Band 4 as UCH37. The specific antibodies poorly recognized the HA-Ub-VS-bound DUBs, so primarily unmodified forms were detected. HA-positive bands 1 and 5 could not be identified with specific antibodies. The relative molecular weight of Band 1 is approximately ~140 kDa and antibodies against USP7 were tried. Band 5 is approximately 18 kDA and is potentially an HA-Ub-VS dimer.

Article Snippet: Proteins were transferred to either nitrocellulose (Protran; VWR) or PVDF (Immobilin FL; EMD Millipore) membranes and immunoblotting was performed using the following antibodies per manufacturer’s instructions: rabbit anti ubiquitin (VU-1) (LifeSensors); rabbit anti PSMA6, rabbit anti USP14, rabbit anti PSMD11 (Bethyl Laboratories); rabbit anti UCH37 (Abcam); rabbit anti USP5, rabbit anti PSMA7, rabbit anti PSMB5, rabbit anti RPT4, rabbit anti GADD34, mouse anti BAX (ProteinTech); goat anti USP14 (E-17), rabbit anti NRF1 (H-285), rabbit anti NRF2 (C-20), goat anti PSMC1 (C-16), rabbit anti ubiquitin (FL-76), rabbit anti PA200 (A-13), rabbit anti PA28beta (L-19) (Santa Cruz); rabbit anti ADRM1, mouse anti eIF2alpha, rabbit anti-phospho eIF2alpha, rabbit anti K48-ubiquitin, rabbit anti K63-ubiquitin (Cell Signaling); mouse anti α1234567 (MCP), mouse anti RPT5, mouse anti RPT1, rabbit anti RPN6 (Enzo); rabbit anti GRP78 (Novus); mouse anti PA28gamma, mouse anti VCP (BD Biosciences); mouse anti HA (Covance); chicken anti P0 (Aves).

Techniques: Incubation, Modification, Activity Assay, Western Blot, Molecular Weight

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 is downregulated after myocardial injury A, RNA transcriptome sequencing( GSE114695 ) was used to reveal the expression profile of USPs (ubiquitin-specific proteases) in myocardial infarction (MI)-induced myocardial injury. RNA transcriptome sequencing was performed on control (n = 3) and 1 d after MI (n = 3) mice heart samples, respectively. We use log2 of the fold change as the source of data for the x axis and −log10 of the P as the source of data for the y axis. Log2 FC >1 and P < 0.05 indicate statistically significant differences. Red and blue points represent the upregulated USPs and the downregulated USPs compared with control group. Meanwhile, black points represent the USPs with no statistical difference compared with the control group. B, Real-time qPCR analysis of the mRNA expression of partial USPs in heart tissues 1 day after MI. C and D, Representative immunoblotting images and quantification showing the protein expression levels of USP13 in the infarct border zone of mouse heart tissues at various time post-myocardial infarction (MI) (n = 7 for each group). E, Uniform manifold approximation and projection (UMAP) plot showing 11 annotated cell types based on the top enriched marker genes from the single-nucleus RNA sequencing (snRNA-seq) data of healthy human hearts. F, Feature plot depicting USP13 scaled expression across all cell clusters in the dataset shown in panel E. Purple dots represent cells expressing USP13 at up to 95 % of the maximum expression level. The top 5 % of high-expressing cells were filtered to avoid any unknown biases. G, Violin plot displaying USP13 scaled expression across all cell types in the heart snRNA-seq data, as shown in panel F. H and I, Representative immunoblotting images and quantification showing the expression levels of USP13 in neonatal mouse ventricular myocytes (NMCMs) and non-NMCMs (n = 4 for each group). J and K, Representative immunoblotting images and quantification showing the expression levels of USP13 in NMCMs undergoing 12 and 24 h of oxygen-glucose deprivation (OGD), (n = 4 for each group). Data were expressed as mean ± SEM. Data in D was analyzed by one-way ANOVA, data in B and I were analyzed by unpaired t -test, data in K were analyzed by Kruskal-Walli's test. ns indicates not significant. ∗P < 0.001. ∗∗P < 0.001. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Sequencing, Expressing, Ubiquitin Proteomics, Control, Western Blot, Marker, RNA Sequencing

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 deficiency exacerbated cardiac injuries and following remodeling process following MI A, Schematic illustration of experimental design. B and C, the serum levels of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) 1 day after MI were detected by commercial kits (n = 6 for each group). D, Representative M-mode echocardiograms obtained from AAV9-Ctrl and AAV9-shUSP13 mice 28d after MI or sham surgery. E and F echocardiographic measurements of left ventricular ejection fraction (EF), fractional shortening (FS) (n = 6 for each group). G and H, Masson trichrome staining of serial sections of mice heart 28 d after MI and the quantified size of scar size (the blue collagen region) (scale bar, 1 mm). Average scar sizes calculated by ImageJ Pro Plus (n = 6 for each group). Data were expressed as mean ± SEM. All the data were analyzed by two-way ANOVA. ns indicates not significant. ∗P < 0.001. ∗∗P < 0.001. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Staining

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 directly interacts with ALDOA A, Schematic illustration for USP13 substrate screening. B, Proteins with high confidence that may interact with USP13 from MS analysis. C, Coimmunoprecipitation (CoIP) of USP13 and ALDOA in NMCMs. D, Representative western blotting for ALDOA in the infarct border zone of mouse heart tissues at various time post-MI. E, CoIP of USP13 and ALDOA in adult mouse heart. F and G, CoIP with anti-Flag or anti-Ha antibodies in 293T cells transfected with Flag-USP13 and Ha-ALDOA. H, Colocalization of Flag-USP13 (green) and Ha-ALDOA (red) in 293T cells. Scale bar = 10 μm. I, Predicted docking module of USP13 and ALDOA visualized by PYMOL. Purple represents USP13, cyan represents ALDOA, yellow is hydrogen bond, and red is the binding site corresponding to USP13. J, Schematic illustration of the USP13 domain deletion construct used in K. K, CoIP with anti-Ha antibody in 293T cells transfected with Flag-USP13-WT, Flag-USP13-Mut-1, Flag-USP13- Mut-2 and Ha-ALDOA.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Western Blot, Transfection, Binding Assay, Construct

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 inhibits MI-induced ferroptosis A and B, Volcano plot and gene set enrichment analysis (GSEA) for differentially expressed genes (DEGs) between nc and si in NMCMs after Oxygen-glucose deprivation (OGD) 24 h. C and D, Representative immunoblotting images and quantification showing the protein expression levels of SLC7A11 and GPX4 in the infarct border zone of mouse heart tissues at 1 day following MI (n = 6 for each group). E, The mRNA expression levels of PTGS2 in the infarct border zone of mouse heart tissues at 1 day following MI (n = 6 for each group). F, The serum levels of malondialdehyde (MDA) 1 day after MI were detected by commercial kits (n = 6 for each group). G and H, Representative immunoblotting images and quantification showing the protein expression levels of SLC7A11 and GPX4 in NMCMs after 24 h of OGD (n = 3 for each group). I and J, Representative fluorescent images and quantification of C11-BODIPY probe (n = 6 for each group), scale bar = 100 μm. The green fluorescence indicated oxidized lipid and the red fluorescence indicated non-oxidized lipid. The green and red ratios were quantitatively analyzed in each group. Data were expressed as mean ± SEM. All the data were analyzed by two-way ANOVA. ns indicates not significant. ∗P < 0.001. ∗∗P < 0.001. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Western Blot, Expressing, Fluorescence

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 restrains ferroptosis post-MI by stabilizing ALDOA A, USP13 and ALDOA protein expression level in the heart tissues collected from control patients and patients with myocardial infarction (n = 3). B and C, Representative immunoblotting images and quantification showing the protein expression levels of ALDOA and USP13 in mouse heart tissues at 1 day following MI (n = 6 for each group). D and E, Representative immunoblotting images and quantification showing the protein expression levels of ALDOA and USP13 in NMCMs after 24 h of OGD (n = 3 for each group). F and G, Representative immunoblotting images and quantification showing the protein expression levels of SLC7A11 and GPX4 in NMCMs after 24 h of OGD (n = 3 for each group). H, The mRNA expression levels of PTGS2 in NMCMs after 24 h of OGD (n = 3 for each group). I and J, the levels of LDH and MDA in H9C2 cells after 8 h of OGD were detected by commercial kits (n = 6 for each group). K and L, Representative fluorescent images and quantification of C11-BODIPY probe (n = 6 for each group), scale bar = 100 μm. The green fluorescence indicated oxidized lipid and the red fluorescence indicated non-oxidized lipid. The green and red ratios were quantitatively analyzed in each group. Data were expressed as mean ± SEM. All the data were analyzed by two-way ANOVA. ns indicates not significant. ∗P < 0.001. ∗∗P < 0.001. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Expressing, Control, Western Blot, Fluorescence

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 modulates the stability of ALDOA through deubiquitination A, Representative western blotting for Flag-USP13 and Ha-ALDOA in 293T cells transfected with Flag-USP13 with various concentrations and Ha-ALDOA. B, The mRNA expression levels of USP13 and ALDOA of A. C, Representative western blotting for Ha-ALDOA in 293T cells transfected with Flag-USP13 and Ha-ALDOA, incubated with the lysosome inhibitor CQ or the proteasome inhibitor MG132 for 6 h. D and E, Representative immunoblotting images and quantification showing the protein expression levels of Ha-ALDOA in 293T cells transfected with Flag-USP13 and Ha-ALDOA and then subjected to CHX for the indicated times (n = 3 for each group). F and G, Representative immunoblotting images and quantification showing the protein expression levels of ALDOA in 293T cells transfected with Si-Ctrl and Si-USP13 and then subjected to CHX for the indicated times (n = 3 for each group). H, Immunoprecipitation with anti-Ha antibody in 293T cells transfected with Flag-USP13, Ha-ALDOA and Myc-Ub and then subjected to MG132 (10 μM). I, Immunoprecipitation with anti-Ha antibody in 293T cells transfected with Flag-USP13, Ha-ALDOA, Myc-Ub, Myc-Ub-K48, and Myc-Ub-K63 and then subjected to MG132 (10 μM). J, K, Immunoprecipitation with anti- Flag or anti-Ha antibodies in 293T cells transfected with Flag-USP13, Ha-ALDOA WT , Ha-ALDOA K13R , Ha-ALDOA K87R and Ha-ALDOA K139R and then subjected to MG132 (10 μM). K, Sequence comparison of ALDOA at K13 among HUMAN, BOVIN, RABIT, MOUSE, RAT. Data were expressed as mean ± SEM. All the data were analyzed by one-way ANOVA. ns indicates not significant. ∗P < 0.001. ∗∗P < 0.001. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Western Blot, Transfection, Expressing, Incubation, Immunoprecipitation, Sequencing, Comparison

Journal: Redox Biology

Article Title: USP13 ameliorates myocardial infarction injury by inhibiting ferroptosis via stabilizing ALDOA

doi: 10.1016/j.redox.2025.103995

Figure Lengend Snippet: USP13 overexpression attenuates cardiac injuries after MI A, Schematic illustration of experimental design. B and C, Representative immunoblotting images and quantification showing the protein expression levels of SLC7A11 and GPX4 in the infarct border zone of mouse heart tissues at 1 day following MI (n = 6 for each group). D, The mRNA expression levels of PTGS2 in the infarct border zone of mouse heart tissues at 1 day following MI (n = 6 for each group). E, F and G, The serum levels of MDA, LDH and CK-MB 1 day after MI were detected by commercial kits (n = 6 for each group). H, Representative M-mode echocardiograms obtained from AAV9-Ctrl and AAV9-USP13 oe mice 28d after MI or sham surgery. I and J echocardiographic measurements of left ventricular ejection fraction (EF), fractional shortening (FS) (n = 6 for each group). K and L, Masson trichrome staining of serial sections of mice heart 28 d after MI and the quantified size of scar size (the blue collagen region) (scale bar, 1 mm). Average scar sizes calculated by ImageJ Pro Plus (n = 6 for each group). Data were expressed as mean ± SEM. All the data were analyzed by two-way ANOVA. ns indicates not significant. ∗P < 0.001. ∗∗P < 0.001. ∗∗∗P < 0.001. ∗∗∗∗P < 0.0001.

Article Snippet: The antibodies are shown below: USP13 (16840-1-AP; Proteintech), USP40 (YT4838, immunoway), USP46 (YN1475, immunoway), β-ACTIN (9470S; Cell Signaling Technology), cardiac troponin I (ab47003; Abcam), SLC7A11(A2413, Abclonal), GPX4(ET1706-45; HUABIO), ALDOA(11217-1-AP; Proteintech), SORBS2(24643-1-AP; Proteintech), TPM2(11038-1-AP; Proteintech), TPM1(A8723; Abclonal), IgG (2729S; Cell Signaling Technology), FLAG (14793S; Cell Signaling Technology), HA (3724S; Cell Signaling Technology), MYC (2278T; Cell Signaling Technology), Cleaved Caspase-3 (9661T, Cell Signaling Technology).

Techniques: Over Expression, Western Blot, Expressing, Staining

Journal: The Kaohsiung journal of medical sciences

Article Title: Ubiquitin specific peptidase 49 inhibits non-small cell lung cancer cell growth by suppressing PI3K/AKT signaling.

doi: 10.1002/kjm2.12073

Figure Lengend Snippet: FIGURE 1 USP49 is lowly expressed in NSCLC, and predicts positive index for NSCLC patients. A, The expression levels of USP49 in normal or cancerous tissues from lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) were analyzed by GEPIA online, which was based on the TCGA database (http://gepia.cancer-pku.cn). B, Twenty pairs of fresh primary NSCLC tissues and individual normal para-cancerous tissues were prepared for qRT-PCR against USP49. GAPDH was used as an internal control. *P < .05, **P < .01. C, Three representative fresh primary NSCLC tissues and individual normal tissues were prepared for immunoblotting against USP49 and β-actin. D, Four NSCLC cell lines and one human bronchial epithelial cells were prepared for immunoblotting against USP49 and β-actin. E, The overall survival periods of NSCLC patients were estimated by Kaplan-Meier plotter (http://kmplot.com). NSCLC, non-small cell lung cancer [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Anti-USP49 antibody was purchased from Proteintech Group (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: The Kaohsiung journal of medical sciences

Article Title: Ubiquitin specific peptidase 49 inhibits non-small cell lung cancer cell growth by suppressing PI3K/AKT signaling.

doi: 10.1002/kjm2.12073

Figure Lengend Snippet: FIGURE 2 Overexpression of USP49 inhibits cell growth by suppressing cell cycle progression in NSCLC cells. A. H322 cells were stably infected with lentiviral PLVX-NC or PLVX-USP49 for indicated time, followed by CCK-8 assay at day 0, 2, 4, and 6. The expression level of USP49 was also detected by immunoblotting. B, H1792 cells were stably infected with lentiviral PLVX-NC or PLVX-USP49 for indicated time, followed by CCK-8 assay at day 0, 2, 4, and 6. The expression level of USP49 was also detected by immunoblotting. C, H322 and H1792 cells were infected with PLVX-NC or PLVX-USP49 for 4 days, and then they were lysed for immunoblotting against Cyclin D1, p53, and USP49. β-actin was used as a loading control. D, H322 cells were infected with PLVX-NC or PLVX-USP49 for 4 days, followed by cell cycle analysis on a flow cytometer. NSCLC, non-small cell lung cancer

Article Snippet: Anti-USP49 antibody was purchased from Proteintech Group (Wuhan, China).

Techniques: Over Expression, Stable Transfection, Infection, CCK-8 Assay, Expressing, Western Blot, Control, Cell Cycle Assay, Flow Cytometry

Journal: The Kaohsiung journal of medical sciences

Article Title: Ubiquitin specific peptidase 49 inhibits non-small cell lung cancer cell growth by suppressing PI3K/AKT signaling.

doi: 10.1002/kjm2.12073

Figure Lengend Snippet: FIGURE 3 USP49 regulates PI3K/AKT signaling in NSCLC cells. A, H322 cells were transfected with empty vector (EV) or Myc-USP49 plasmids for 72 hours, followed by immunoblotting against p-p85, PI3K p85, PTEN, and Myc. β-actin was used as a loading control. B, H322 cells transfected with EV or Myc-USP49 plasmids were also prepared for immunoblotting against p-AKT, AKT, p-mTOR, mTOR, Myc and β-actin. C, H322 cells were infected with PLVX-NC or PLVX-USP49 for 4 days, followed by immunoblotting against p-p85, PI3K p85, PTEN, USP49, and β-actin. D, Above cells were also prepared for immunoblotting against p-AKT, AKT, p-mTOR, mTOR, USP49, and β-actin. NSCLC, non-small cell lung cancer

Article Snippet: Anti-USP49 antibody was purchased from Proteintech Group (Wuhan, China).

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Infection

Journal: The Kaohsiung journal of medical sciences

Article Title: Ubiquitin specific peptidase 49 inhibits non-small cell lung cancer cell growth by suppressing PI3K/AKT signaling.

doi: 10.1002/kjm2.12073

Figure Lengend Snippet: FIGURE 4 USP49 deubiquitinates and stabilizes PTEN in NSCLC cells. A, The correlation analysis between PTEN and USP49 was retrieved from GEPIA database (http://gepia.cancer-pku.cn). B, HEK293T cells were transfected with Myc-USP49 or Flag-PTEN along with HA-Ub plasmids. Forty-eight hours later, cells were lysed for immunoprecipitation against Flag, followed by immunoblotting against HA and Flag. C, Above whole cell lysates were also prepared for immunoblotting against Flag, Myc, and β-actin. D, H322 cells were transfected with Myc-USP49 or EV for 24 hours, followed by CHX chase assay at indicated time. Then, immunoblotting was carried out to assess the expression levels of PTEN and Myc-USP49. β-actin was used as a loading control. E, Statistically analysis of figure D. NSCLC, non-small cell lung cancer

Article Snippet: Anti-USP49 antibody was purchased from Proteintech Group (Wuhan, China).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Clinical and immunological characterization of a Netherton syndrome infant with a large SPINK gene cluster deletion and a c.1258A>G polymorphism in SPINK5

doi: 10.3389/fimmu.2025.1658444

Figure Lengend Snippet: Genetic and protein expression analysis. (A) Whole-exome sequencing identified a heterozygous deletion spanning the SPINK gene cluster, including SPINK5 . (B) Sanger sequencing of the patient and his mother revealed a polymorphism in SPINK5 ( NM_006846 ; exon 14, c.1258A>G, p.K420E). (C) LEKTI and KLK5 expression in peripheral blood mononuclear cells (PBMCs) from the patient compared to healthy controls (HC, n=3). (D) KLK5 level in the patient’s plasma (seven time points from initial hospitalization) compared with that in healthy controls (HC, n = 8).

Article Snippet: Membranes were blocked in 5% skim milk and incubated overnight at 4°C with primary antibodies against LEKTI (29808-1-AP, Proteintech, USA), antibodies against KLK5 (38528, SAB, USA) and HRP-conjugated β-actin recombinant rabbit monoclonal antibody (ET1702-67, HUABIO, China).

Techniques: Expressing, Sequencing, Clinical Proteomics