Journal: Journal of Biological Chemistry

Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex

doi: 10.1074/jbc.ra118.006290

Figure Lengend Snippet: Figure 4. Nrf1 interacts with the TIP60 complex. (A) Wild-type (WT) and Nrf1-/- NIH-3T3 cell lines were treated with 200 nM carfilzomib (CFZ) for 8 hours. The cells were then subjected to chromatin immunoprecipitation (ChIP) with one of IgG, Nrf1, RUVBL1, or TIP60 antibodies. These samples were then analyzed by quantitative PCR with primers specific for antioxidant response element (ARE)- containing promoter regions of proteasome genes PSMA7, PSMB7, and PSMD12. Error bars denote SD (n=3). (B) HEK293 cells stably expressing tagged Nrf1 (Nrf13xFLAG) were treated or not with 200 nM CFZ for 8 hours. The cell lysates were then subjected to immunoprecipitation with anti-FLAG beads and analyzed by immunoblotting with antibodies specific for FLAG, RUVBL1, and RUVBL2. The lysate lanes were loaded with 5% of the input that was used for immunoprecipitation. The experiments were performed three independent times and a representative blot is shown.

Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Actinomycin D and bortezomib disrupt protein homeostasis in Wilms tumor

doi: 10.1101/2024.06.11.598518

Figure Lengend Snippet: (A) Western blots for ribosomal protein subunits RPL5, RPL7, RPL11, and RPL26 in WiT49 and 17.94 following 48-hour treatment of DMSO, 2nM actD, 8nM BTZ, or 2nM ActD + 8nM BTZ. (B) Protein levels of proteasome subunits PSMA6 (α1), PSMB1 (β6), PSMB2 (β4), PSMB5 (β5), and PSMD1 (P112), and POMP in WiT49 and 17.94 following 18-, 24-, 48-, and 72-hour 2nM actD treatment versus vehicle (DMSO). (C) Quantification of relative proteasome-specific chymotrypsin-like activity in WiT49 and 17.94 cells following 24 hours of DMSO versus 2nM actD, 8nM BTZ, or 2nM actD + 8nM BTZ treatments (Student’s t-test p-value versus vehicle: *<0.05, ** <0.01, ****<0.0001).

Article Snippet: Primary antibodies used are as follows and were diluted to 1:3,000 unless otherwise stated: Tubulin (Cell Signaling Technology 3873, RRID:AB_1904178), GAPDH (Cell Signaling Technology 97166, RRID:AB_2756824), Caspase-3 (Cell Signaling Technology 9665, RRID:AB_2069872), cleaved Caspase-3 (Cell Signaling Technology 9664, RRID:AB_2070042; 1:1,000), Caspase-7 (Cell Signaling Technology 12827, RRID:AB_2687912), cleaved Caspase-7 (Cell Signaling Technology 8438, RRID:AB_11178377; 1:1,000), Caspase-9 (Cell Signaling Technology 9508, RRID:AB_2068620), cleaved Caspase-9 (Cell Signaling Technology 7237, RRID:AB_10895832; 1:1,000), Cyclin D2 (Cell Signaling Technology 3741; RRID:AB_2070685), CDT1 (Cell Signaling Technology 8064, RRID:AB_10896851), Cyclin E1 Cell Signaling Technology 20808, RRID:AB_2783554), Cyclin A2 (Cell Signaling Technology 91500RRID:AB_3096041), PARP (Cell Signaling Technology 9532, RRID:AB_659884), cleaved PARP (Cell Signaling Technology 5625, RRID:AB_10699459; 1:1,000), POMP (Cell Signaling Technology 15141, RRID:AB_2798726), PSMA6 (Cell Signaling Technology 2459, RRID:AB_2268879), PSMB1 (Invitrogen PA5-49648, RRID: AB_2635102; 1:5,000), PSMB2 (Proteintech 15154-1-AP, RRID:AB_2300322; 1:5,000), PSMB5 (Cell Signaling Technology 12919, RRID:AB_2798061; 1:5,000), PSMD1 (Sigma SAB2104781, RRID:AB_10668741), RPL5 (Cell Signaling Techonology 51345, RRID:AB_2799391), RPL7 (abcam ab72550, RRID:AB_1270391), RPL11 (Cell Signaling Technology 18163, RRID:AB_2798794), RPL26 (Cell Signaling Technology 2065, RRID:AB_2146242), Total AKT (Cell Signaling Technology 9272, RRID:AB_329827), Phospho-AKT (Ser473) (Cell Signaling Technology 4060, RRID:AB_2315049; 1:1,000), Total 4E-BP1 (Cell Signaling Technology 9452, RRID:AB_331692), Phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology 2855, RRID:AB_560835; 1:1,000), Total P70-S6K1 (Cell Signaling Technology 34475, RRID:AB_2943679), and Phospho-P70-S6K1 (Thr389) (Invitrogen 710095, AB_2532559; 1:1,000).

Techniques: Western Blot, Activity Assay

Journal: bioRxiv

Article Title: Actinomycin D and bortezomib disrupt protein homeostasis in Wilms tumor

doi: 10.1101/2024.06.11.598518

Figure Lengend Snippet: (A) Top KEGG pathways enriched in RNA-seq of DAWT versus FHWT. (B) Top reactome pathways enriched in RNA-seq of DAWT versus FHWT. (C,D) Overall survival of tumors stratified according to expression of proteasome enzymatic subunit genes PSMB1, PSMB2, PSMB5 among patients with favorable histology tumors (C) or anaplastic histology (D) . (Log Rank test p-value: *<0.05, ** 0.01)

Article Snippet: Primary antibodies used are as follows and were diluted to 1:3,000 unless otherwise stated: Tubulin (Cell Signaling Technology 3873, RRID:AB_1904178), GAPDH (Cell Signaling Technology 97166, RRID:AB_2756824), Caspase-3 (Cell Signaling Technology 9665, RRID:AB_2069872), cleaved Caspase-3 (Cell Signaling Technology 9664, RRID:AB_2070042; 1:1,000), Caspase-7 (Cell Signaling Technology 12827, RRID:AB_2687912), cleaved Caspase-7 (Cell Signaling Technology 8438, RRID:AB_11178377; 1:1,000), Caspase-9 (Cell Signaling Technology 9508, RRID:AB_2068620), cleaved Caspase-9 (Cell Signaling Technology 7237, RRID:AB_10895832; 1:1,000), Cyclin D2 (Cell Signaling Technology 3741; RRID:AB_2070685), CDT1 (Cell Signaling Technology 8064, RRID:AB_10896851), Cyclin E1 Cell Signaling Technology 20808, RRID:AB_2783554), Cyclin A2 (Cell Signaling Technology 91500RRID:AB_3096041), PARP (Cell Signaling Technology 9532, RRID:AB_659884), cleaved PARP (Cell Signaling Technology 5625, RRID:AB_10699459; 1:1,000), POMP (Cell Signaling Technology 15141, RRID:AB_2798726), PSMA6 (Cell Signaling Technology 2459, RRID:AB_2268879), PSMB1 (Invitrogen PA5-49648, RRID: AB_2635102; 1:5,000), PSMB2 (Proteintech 15154-1-AP, RRID:AB_2300322; 1:5,000), PSMB5 (Cell Signaling Technology 12919, RRID:AB_2798061; 1:5,000), PSMD1 (Sigma SAB2104781, RRID:AB_10668741), RPL5 (Cell Signaling Techonology 51345, RRID:AB_2799391), RPL7 (abcam ab72550, RRID:AB_1270391), RPL11 (Cell Signaling Technology 18163, RRID:AB_2798794), RPL26 (Cell Signaling Technology 2065, RRID:AB_2146242), Total AKT (Cell Signaling Technology 9272, RRID:AB_329827), Phospho-AKT (Ser473) (Cell Signaling Technology 4060, RRID:AB_2315049; 1:1,000), Total 4E-BP1 (Cell Signaling Technology 9452, RRID:AB_331692), Phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology 2855, RRID:AB_560835; 1:1,000), Total P70-S6K1 (Cell Signaling Technology 34475, RRID:AB_2943679), and Phospho-P70-S6K1 (Thr389) (Invitrogen 710095, AB_2532559; 1:1,000).

Techniques: RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: Actinomycin D and bortezomib disrupt protein homeostasis in Wilms tumor

doi: 10.1101/2024.06.11.598518

Figure Lengend Snippet: (A,B) GSEA plot showing enrichment for KEGG_PROTEASOME (A) and REACTOME_APC_C_MEDIATED_DEGRADATION_OF_CELL_CYCLE_PROTEINS (B) in anaplastic Wilms tumor vs. favorable-histology Wilms tumors in TARGET dataset. (C) Overall survival of Wilms tumors, regardless of histology, stratified according to expression of proteasome enzymatic subunit genes PSMB1, PSMB2, PSMB5 among patients (Log rank test p-value: *<0.05, *** 0.001). (D) Model for effect of actinomycin D on protein homeostasis in Wilms tumor cells.

Article Snippet: Primary antibodies used are as follows and were diluted to 1:3,000 unless otherwise stated: Tubulin (Cell Signaling Technology 3873, RRID:AB_1904178), GAPDH (Cell Signaling Technology 97166, RRID:AB_2756824), Caspase-3 (Cell Signaling Technology 9665, RRID:AB_2069872), cleaved Caspase-3 (Cell Signaling Technology 9664, RRID:AB_2070042; 1:1,000), Caspase-7 (Cell Signaling Technology 12827, RRID:AB_2687912), cleaved Caspase-7 (Cell Signaling Technology 8438, RRID:AB_11178377; 1:1,000), Caspase-9 (Cell Signaling Technology 9508, RRID:AB_2068620), cleaved Caspase-9 (Cell Signaling Technology 7237, RRID:AB_10895832; 1:1,000), Cyclin D2 (Cell Signaling Technology 3741; RRID:AB_2070685), CDT1 (Cell Signaling Technology 8064, RRID:AB_10896851), Cyclin E1 Cell Signaling Technology 20808, RRID:AB_2783554), Cyclin A2 (Cell Signaling Technology 91500RRID:AB_3096041), PARP (Cell Signaling Technology 9532, RRID:AB_659884), cleaved PARP (Cell Signaling Technology 5625, RRID:AB_10699459; 1:1,000), POMP (Cell Signaling Technology 15141, RRID:AB_2798726), PSMA6 (Cell Signaling Technology 2459, RRID:AB_2268879), PSMB1 (Invitrogen PA5-49648, RRID: AB_2635102; 1:5,000), PSMB2 (Proteintech 15154-1-AP, RRID:AB_2300322; 1:5,000), PSMB5 (Cell Signaling Technology 12919, RRID:AB_2798061; 1:5,000), PSMD1 (Sigma SAB2104781, RRID:AB_10668741), RPL5 (Cell Signaling Techonology 51345, RRID:AB_2799391), RPL7 (abcam ab72550, RRID:AB_1270391), RPL11 (Cell Signaling Technology 18163, RRID:AB_2798794), RPL26 (Cell Signaling Technology 2065, RRID:AB_2146242), Total AKT (Cell Signaling Technology 9272, RRID:AB_329827), Phospho-AKT (Ser473) (Cell Signaling Technology 4060, RRID:AB_2315049; 1:1,000), Total 4E-BP1 (Cell Signaling Technology 9452, RRID:AB_331692), Phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology 2855, RRID:AB_560835; 1:1,000), Total P70-S6K1 (Cell Signaling Technology 34475, RRID:AB_2943679), and Phospho-P70-S6K1 (Thr389) (Invitrogen 710095, AB_2532559; 1:1,000).

Techniques: Wilms Tumor Assay, Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: JAK-centric explainable few-shot gene-expression diagnosis framework for alopecia via MultiPLIER priors and relation-style set-to-set comparison

doi: 10.3389/fmolb.2025.1753206

Figure Lengend Snippet: RT-qPCR validation of the IL2RB/IL2RG–EOMES–GZMA cytotoxic/JAK axis in alopecia areata (AA). Relative mRNA levels of EOMES, GZMA, IL2RB, and IL2RG are significantly elevated in AA lesional scalp compared with healthy controls, whereas androgenetic alopecia (AGA) samples are comparable to controls, indicating that this cytotoxic/JAK module is transcriptionally active in AA but largely quiescent in AGA. Data are shown as mean ± SD. Statistical annotations: ** p < 0.01 vs. control; n.s., not significant (AGA vs. control).

Article Snippet: Equal amounts of protein were resolved by SDS–PAGE and transferred to PVDF membranes at 200 mA for 2 h. Membranes were blocked in 5% non-fat milk at room temperature for 2 h and incubated overnight at 4 ° C with primary antibodies against β -actin (1:4000; 20536-1-AP, Proteintech, China), EOMES (1:5000; 83945-5-RR, Proteintech, China), GZMA (1:500; 11288-1-AP, Proteintech, China), IL2RB (1:5000; 13602-1-AP, Proteintech, China), and IL2RG (1:500; 11409-1-AP, Proteintech, China).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Control

Journal: Frontiers in Molecular Biosciences

Article Title: JAK-centric explainable few-shot gene-expression diagnosis framework for alopecia via MultiPLIER priors and relation-style set-to-set comparison

doi: 10.3389/fmolb.2025.1753206

Figure Lengend Snippet: GSEA plots for key genes. (a) EOMES. (b) GZMA. (c) IL2RB. (d) IL2RG.

Article Snippet: Equal amounts of protein were resolved by SDS–PAGE and transferred to PVDF membranes at 200 mA for 2 h. Membranes were blocked in 5% non-fat milk at room temperature for 2 h and incubated overnight at 4 ° C with primary antibodies against β -actin (1:4000; 20536-1-AP, Proteintech, China), EOMES (1:5000; 83945-5-RR, Proteintech, China), GZMA (1:500; 11288-1-AP, Proteintech, China), IL2RB (1:5000; 13602-1-AP, Proteintech, China), and IL2RG (1:500; 11409-1-AP, Proteintech, China).

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: JAK-centric explainable few-shot gene-expression diagnosis framework for alopecia via MultiPLIER priors and relation-style set-to-set comparison

doi: 10.3389/fmolb.2025.1753206

Figure Lengend Snippet: Western blot validation of the IL2RB/IL2RG–EOMES–GZMA cytotoxic/JAK axis at the protein level. Protein expression of EOMES, GZMA, IL2RB, and IL2RG is markedly upregulated in AA lesional scalp compared with healthy controls, concordant with the RT-qPCR results, whereas AGA-affected scalp shows no significant difference relative to controls. Representative immunoblots and densitometric quantification (normalized to β -actin) are shown. Data are presented as mean ± SD. Statistical annotations: ** p < 0.01 vs. control; n.s., not significant (AGA vs. control).

Article Snippet: Equal amounts of protein were resolved by SDS–PAGE and transferred to PVDF membranes at 200 mA for 2 h. Membranes were blocked in 5% non-fat milk at room temperature for 2 h and incubated overnight at 4 ° C with primary antibodies against β -actin (1:4000; 20536-1-AP, Proteintech, China), EOMES (1:5000; 83945-5-RR, Proteintech, China), GZMA (1:500; 11288-1-AP, Proteintech, China), IL2RB (1:5000; 13602-1-AP, Proteintech, China), and IL2RG (1:500; 11409-1-AP, Proteintech, China).

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

Journal: Food Science and Human Wellness

Article Title: Sucrose-free hawthorn leathers formulated with fructooligosaccharides and xylooligosaccharides ameliorate high-fat diet induced inflammation, glucose and lipid metabolism in liver of mice

doi: 10.1016/j.fshw.2022.03.033

Figure Lengend Snippet: Fig. 4 Effect of hawthorn leather treatment on expression levels of inflammatory cytokines and NF-κB signaling pathway in the liver tissue of mice. mRNA expression level of (A) IL-1β, (B) Nos2, (C) Cox2, (D) TLR4, (E) MyD88, and (F) NF-κB; (G) Images of western blotting; Protein expression ratio of (H) MyD88 to GAPDH and (I) NF-κB to GAPDH. Data are expressed as mean ± SD (8 mice/group). Values of each group with different letters over the bars are significantly different (P < 0.05) by analysis of one-way ANOVA followed by Tukey’s multiple range test.

Article Snippet: Then, the membrane was incubated with specific primary antibody (1 : 1 000) against MyD88 (CST, cat. number 4283S), NF-κB (p65) (CST, cat. number 8242S), PI3K (p110β) (Proteintech, cat. number 20584-1-AP), AKT (Proteintech, cat. number 10176-2-AP), Thr308 phosphorylated AKT (p-AKT) (CST, cat. number 13038T), mTOR (Proteintech, cat. number 20657-1-AP), Ser2448 phosphorylated mTOR (p-mTOR) (CST, cat. number 5536T), PPAR-γ (CST, cat. number 2435T), FASN (CST, cat. number 3180T), and HSL (CST, cat. number 18381T) overnight at 4 °C, respectively.

Techniques: Expressing, Western Blot

Journal: International journal of biological macromolecules

Article Title: Aberrant serum-derived FN1 variants bind to integrin β1 on glomerular endothelial cells contributing to thin basement membrane nephropathy.

doi: 10.1016/j.ijbiomac.2024.136282

Figure Lengend Snippet: Fig. 5. Expression changes of key GBM proteins and their response to FN1 Variants in TBMN. Immunofluorescence experiments assessed the expression of Laminin α5β2, COL4A3/4/5 and Integrin β1 in the GBM. The findings revealed non-homogeneous linear structure expression of these key GBM proteins in the presence of FN1 variants, indicating their potential involvement in the pathogenesis of TBMN. HC = healthy control.

Article Snippet: Incubating primary antibodies overnight at 4 ◦C, with specific primary antibodies against HA (1:2000, ab9110, Abcam), Integrin β1 (1:1000, 12,594–1-AP, Proteintech), β-tubulin (1:3000, ab0039, Abways), Laminin α5 (1:1000, ab210957, Abcam), Laminin β2 (1:1000, ab210956, Abcam), Laminin γ1 (1:1000, ab233389, Abcam) in Tris-Buffered Saline Tween-20 (TBST) containing 5 % skim milk.

Techniques: Expressing, Immunofluorescence, Control

Journal: International journal of biological macromolecules

Article Title: Aberrant serum-derived FN1 variants bind to integrin β1 on glomerular endothelial cells contributing to thin basement membrane nephropathy.

doi: 10.1016/j.ijbiomac.2024.136282

Figure Lengend Snippet: Fig. 6. Abnormal co-localization of FN1 variants with Integrin β1 and its impact on other GBM components. Immunofluorescence studies investigated the rela tionship between FN1 variants and the expression patterns of GBM components. The findings reveal abnormal co-localization of FN1 variants with Integrin β1, accompanied by reduced co-localization of other GBM components. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Incubating primary antibodies overnight at 4 ◦C, with specific primary antibodies against HA (1:2000, ab9110, Abcam), Integrin β1 (1:1000, 12,594–1-AP, Proteintech), β-tubulin (1:3000, ab0039, Abways), Laminin α5 (1:1000, ab210957, Abcam), Laminin β2 (1:1000, ab210956, Abcam), Laminin γ1 (1:1000, ab233389, Abcam) in Tris-Buffered Saline Tween-20 (TBST) containing 5 % skim milk.

Techniques: Immunofluorescence, Expressing

Journal: International journal of biological macromolecules

Article Title: Aberrant serum-derived FN1 variants bind to integrin β1 on glomerular endothelial cells contributing to thin basement membrane nephropathy.

doi: 10.1016/j.ijbiomac.2024.136282

Figure Lengend Snippet: Fig. 7. Competitive Binding of FN1 Variants to Integrin β1. (A and B) Duolink proximity ligation assay and Co-IP were employed to analyze the protein interaction between FN1 variants and Integrin β1. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Incubating primary antibodies overnight at 4 ◦C, with specific primary antibodies against HA (1:2000, ab9110, Abcam), Integrin β1 (1:1000, 12,594–1-AP, Proteintech), β-tubulin (1:3000, ab0039, Abways), Laminin α5 (1:1000, ab210957, Abcam), Laminin β2 (1:1000, ab210956, Abcam), Laminin γ1 (1:1000, ab233389, Abcam) in Tris-Buffered Saline Tween-20 (TBST) containing 5 % skim milk.

Techniques: Binding Assay, Proximity Ligation Assay, Co-Immunoprecipitation Assay