rabbit polyclonal anti nub1 antibody  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis

doi: 10.1038/s41598-026-38420-y

Figure Lengend Snippet: Immunohistochemical staining of NUB1, NEDD8, IL-6, NF-kB in RA and OA synovial tissues. ( a ) Representative images (magnification, ×200) of immunohistochemistry for NUB1, NEDD8, and IL-6 in synovial tissues from patients with RA and OA. Expression of NEDD8 and IL-6 was higher in the intimal lining of RA synovium, and expression of NUB1 was lower compared with OA (see Fig. for quantification). Scale bar = 100 μm, ( b ) Representative images (magnification, ×400) of immunohistochemistry for NUB1 and p65 in synovial tissues from patients with RA and OA. In RA, regions with reduced NUB1 expression showed prominent nuclear localization of p65, whereas in OA, areas with higher NUB1 expression displayed weaker p65 nuclear staining. Negative control (NC) sections were stained with rabbit IgG under identical conditions. Scale bar = 50 μm.

Article Snippet: For immunohistochemistry, anti-NEDD8 (19E3) rabbit mAb (#2754), and anti-p65 rabbit mAb (#8242), were purchased from Cell Signaling Technology, and a rabbit polyclonal anti-NUB1 antibody (#14343-1-AP), and anti-rabbit polyclonal anti-IL-6 antibody (#21865-1-AP) were purchased from Proteintech, and normal Rabbit IgG control (#AB-105-C) was purchased from R༆D).

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry, Expressing, Negative Control

Journal: Scientific Reports

Article Title: Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis

doi: 10.1038/s41598-026-38420-y

Figure Lengend Snippet: Immunohistochemical scoring of NUB1, NEDD8, and IL-6 in RA and OA synovial tissues. Synovial tissue sections from RA (n = 5) and OA (n = 5) patients were immunostained for NUB1, NEDD8, IL-6, and CCL5. For each patient, three randomly selected fields were evaluated in the lining and sublining zones. Staining was semiquantitatively scored as described in Methods. Graphs show the total scores as well as the average scores for the lining and sublining zones. For the total scores, NUB1 was higher in OA than in RA, whereas NEDD8 and IL-6 were significantly higher in RA; these differences were most prominent in the lining layer. Data are presented as mean ± SEM. Statistical analysis was performed using unpaired t test with Welch’s correction or Mann-Whitney U test (* p <0.05; ** p <0.01).

Article Snippet: For immunohistochemistry, anti-NEDD8 (19E3) rabbit mAb (#2754), and anti-p65 rabbit mAb (#8242), were purchased from Cell Signaling Technology, and a rabbit polyclonal anti-NUB1 antibody (#14343-1-AP), and anti-rabbit polyclonal anti-IL-6 antibody (#21865-1-AP) were purchased from Proteintech, and normal Rabbit IgG control (#AB-105-C) was purchased from R༆D).

Techniques: Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis

doi: 10.1038/s41598-026-38420-y

Figure Lengend Snippet: Basal and IL-1-induced expression of NUB1 in RA and OA FLS. ( a ) Left panel: Basal NUB1 mRNA levels in RA and OA FLS were quantified by RT-qPCR and normalized to GAPDH. Right panel: Following stimulation with IL-1β (2 ng/mL) for 6 h, the fold change in NUB1 expression relative to unstimulated cells was calculated for each group (n = 6 each). Basal NUB1 mRNA expression was comparable between RA and OA FLS ( p = 0.153), whereas IL-1β–induced upregulation of NUB1 was significantly lower in RA FLS than in OA FLS. ( b ) NUB1 protein expression in RA and OA FLS following IL-1β stimulation. Left panel: RA and OA FLS were stimulated with IL-1β (2 ng/mL) for 24 h, and NUB1 protein expression was evaluated by Western blotting. Right panel: NUB1 band intensities were quantified after normalization to α-tubulin. IL-1β-induced NUB1 protein expression was also significantly reduced in RA FLS compared with OA FLS. These indicate defective IL-1–mediated NUB1 induction in RA FLS. Data are presented as mean ± SEM. Statistical significance was determined using unpaired t test with Welch’s correction. (** p < 0.01, *** p < 0.001).

Article Snippet: For immunohistochemistry, anti-NEDD8 (19E3) rabbit mAb (#2754), and anti-p65 rabbit mAb (#8242), were purchased from Cell Signaling Technology, and a rabbit polyclonal anti-NUB1 antibody (#14343-1-AP), and anti-rabbit polyclonal anti-IL-6 antibody (#21865-1-AP) were purchased from Proteintech, and normal Rabbit IgG control (#AB-105-C) was purchased from R༆D).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis

doi: 10.1038/s41598-026-38420-y

Figure Lengend Snippet: Regulation of NUB1 expression by MAPKs . RA and OA FLS (n = 5 each) were stimulated with IL-1β (2 ng/mL) for 6h in the presence or absence of MAPK inhibitors: SP600125 (JNK inhibitor), SB203580 (p38 MAPK inhibitor), and U0126 (MEK1/2–ERK inhibitor). NUB1 mRNA expression is shown as fold change relative to IL-1β stimulation alone. ( a ) Quantitative analysis demonstrated that none of the MAPK inhibitors significantly reduced IL-1β–induced NUB1 mRNA expression in either RA or OA FLS (IL-1β vs. IL-1β + SB203580, adjusted p > 0.9999; IL-1β vs. IL-1β + SP600125, adjusted p > 0.9999; IL-1β vs. IL-1β + U0126, adjusted p = 0.8285). ( b ) IL-6 mRNA expression was quantified by RT-qPCR and normalized to GAPDH. IL-6 expression was significantly reduced by each MAPK inhibitor compared with IL-1β stimulation alone (**** p < 0.0001), confirming effective inhibition of MAPK-dependent signaling. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: For immunohistochemistry, anti-NEDD8 (19E3) rabbit mAb (#2754), and anti-p65 rabbit mAb (#8242), were purchased from Cell Signaling Technology, and a rabbit polyclonal anti-NUB1 antibody (#14343-1-AP), and anti-rabbit polyclonal anti-IL-6 antibody (#21865-1-AP) were purchased from Proteintech, and normal Rabbit IgG control (#AB-105-C) was purchased from R༆D).

Techniques: Expressing, Quantitative RT-PCR, Inhibition

Journal: Scientific Reports

Article Title: Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis

doi: 10.1038/s41598-026-38420-y

Figure Lengend Snippet: ( a ) mRNA half life of NUB1. RA and OA FLS (n = 5 each) were stimulated with IL-1β (2 ng/mL) for 6 h, followed by treatment with actinomycin D (10 μg/mL) to block transcription. NUB1 mRNA expression was then measured by qRT-PCR at 0, 4, 8, 12, and 18 h after actinomycin D treatment. Expression levels were normalized to GAPDH and presented as fold change relative to the 0 h time point (i.e., the level observed immediately after 6 h of IL-1β stimulation). Decay curves showed no significant differences between groups. mRNA decay curves were fitted using a one-phase exponential decay model by nonlinear regression to calculate NUB1 mRNA half-life, and decay constants (K) for RA and OA were compared using an extra sum-of-squares F -test. ( b ) Effect of SNHG12 knockdown on NUB1 expression in RA and OA FLS. SNHG12 (Small Nucleolar RNA Host Gene 12), a long non-coding RNA associated with NUB1, was depleted in RA and OA FLS using SNHG12 siRNA (knockdown efficiency >80%). RA and OA (n = 7 each) FLS were stimulated with IL-1β (2 ng/mL) for 6 h, and NUB1 mRNA expression was quantified by RT-qPCR and normalized to GAPDH. Left panel: Fold change in NUB1 expression following IL-1β stimulation in siCT-transfected cells (IL-1/unstimulated). Right panel: Fold change in NUB1 expression following IL-1β stimulation after SNHG12 knockdown (siSNHG12 + IL-1/siSNHG12 unstimulated). Knockdown of SNHG12 had little effect on the RA–OA difference in NUB1 induction. Data are presented as mean ± SEM. Statistical analysis was performed using unpaired t test with Welch’s correction (** p < 0.01, *** p < 0.001). ( c ) NUB1 promoter activity in RA and OA FLS. RA and OA FLS (n = 5 each) were transfected with a luciferase reporter construct containing the NUB1 promoter region and co-transfected with Renilla vector for normalization (see Material and Methods). After transfection, cells were stimulated with IL-1β (2 ng/mL). Firefly luciferase activity was normalized to Renilla and expressed as fold change calculated by dividing the IL-1β–stimulated value by the corresponding unstimulated (media) control at each time point. Limited promoter induction was noted, possibly because transcription requires epigenetic marks or the three-dimensional structure of chromatin not present in plasmids. No significant differences in IL-1β–induced NUB1 promoter activity were observed between RA and OA FLS at 1 h ( p = 0.209), 3 h ( p = 0.415), or 6 h ( p = 0.446). Data are presented as mean ± SEM. Statistical analysis was performed using an unpaired t test with Welch’s correction.

Article Snippet: For immunohistochemistry, anti-NEDD8 (19E3) rabbit mAb (#2754), and anti-p65 rabbit mAb (#8242), were purchased from Cell Signaling Technology, and a rabbit polyclonal anti-NUB1 antibody (#14343-1-AP), and anti-rabbit polyclonal anti-IL-6 antibody (#21865-1-AP) were purchased from Proteintech, and normal Rabbit IgG control (#AB-105-C) was purchased from R༆D).

Techniques: Blocking Assay, Expressing, Quantitative RT-PCR, Knockdown, Transfection, Activity Assay, Luciferase, Construct, Plasmid Preparation, Control

Journal: Scientific Reports

Article Title: Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis

doi: 10.1038/s41598-026-38420-y

Figure Lengend Snippet: Effects of epigenetic inhibitors on IL-1β–induced NUB1 expression in RA and OA fibroblast-like synoviocytes (FLS). RA and OA FLS were pretreated with epigenetic inhibitors and subsequently stimulated with IL-1β (2 ng/mL). NUB1 mRNA expression was quantified by qRT-PCR. Fold change was calculated as the ratio of NUB1 mRNA expression in IL-1β–stimulated cells to the corresponding unstimulated control; specifically, DMSO alone for the IL-1/DMSO condition and the inhibitor-alone control for inhibitor-treated conditions. ( a ) 5-aza-deoxycytidine (5-aza-dC; DNA methyltransferase inhibitor). RA and OA FLS were treated with 5-aza-dC for 14 days prior to stimulation with IL-1β (n = 7 each). 5-aza-dC partially reduced the difference in IL-1β–induced NUB1 expression between RA and OA. Under control conditions, IL-1β–induced NUB1 expression was significantly higher in OA than in RA ( p = 0.024). Following 5-aza-dC treatment, the magnitude of the OA–RA difference was no longer statistically significant ( p = 0.488), indicating partial attenuation of the baseline RA–OA difference. ( b ) EPZ6438 (EZH2 inhibitor; histone methylation inhibitor). RA and OA FLS were treated with EPZ6438 for 12 or 24 hours before IL-1β stimulation (n = 5 each). treatment with EPZ6438 partially reversed the difference in IL-1β–induced NUB1 expression between RA and OA. Under control conditions, IL-1β–induced NUB1 expression was significantly higher in OA than in RA. This difference was no longer significant after EPZ6438 treatment at 12 h ( p = 0.329) or 24 h ( p = 0.512). ( c ) Left panel: ITF2357 (pan-HDAC inhibitor); Right panel: MS275 (HDAC1/3 selective inhibitor). RA and OA FLS were treated with each HDAC inhibitor for 12 or 24 hours, followed by IL-1β stimulation (ITF2357; n = 5 each, MS275; n = 6 each). Under control conditions, IL-1β–induced NUB1 expression was significantly higher in OA than in RA in the ITF2357 panel. Following ITF2357 treatment, the OA–RA difference was no longer statistically significant at 12 h ( p = 0.596) or 24 h ( p = 0.072). Under control conditions, IL-1β–induced NUB1 expression was also significantly higher in OA than in RA in the MS275 panel. Following MS275 treatment, the OA–RA difference was no longer statistically significant at either 12 h ( p = 0.944) or 24 h ( p = 0.846). These data indicate that histone modifications are required for differential induction of NUB1 in OA compared with RA. Circles and squares represent mean fold change for OA and RA FLS, respectively. Statistical analysis was performed at each time point and condition performed by comparing the fold change values between RA and OA FLS using unpaired t test with Welch’s correction (* p < 0.05, ** p < 0.01).

Article Snippet: For immunohistochemistry, anti-NEDD8 (19E3) rabbit mAb (#2754), and anti-p65 rabbit mAb (#8242), were purchased from Cell Signaling Technology, and a rabbit polyclonal anti-NUB1 antibody (#14343-1-AP), and anti-rabbit polyclonal anti-IL-6 antibody (#21865-1-AP) were purchased from Proteintech, and normal Rabbit IgG control (#AB-105-C) was purchased from R༆D).

Techniques: Expressing, Quantitative RT-PCR, Control, Methylation

Journal: Cell Reports Medicine

Article Title: The potential of lazertinib and amivantamab combination therapy as a treatment strategy for uncommon EGFR -mutated NSCLC

doi: 10.1016/j.xcrm.2025.101929

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Phospho-EGFR (Tyr1068) , Cell Signaling Technology , Cat#2234 RRID: AB_331701.

Techniques: Recombinant, Sequencing, Software

Journal: iScience

Article Title: Cbl and Cbl-b ubiquitin ligases are essential for intestinal epithelial stem cell maintenance

doi: 10.1016/j.isci.2024.109912

Figure Lengend Snippet: Conditional Cbl/Cblb deletion impairs self-renewal in intestinal crypt organoid cultures by upregulating the Akt-mTOR pathway Equal number of crypts isolated from Cbl flox/flox ; Cblb flox/flox (FF Control) and Cbl flox/flox ; Cblb flox/flox ; R26cre ERT2 (FF/creERT) mice were plated in 100% Matrigel in the presence of growth factors to form organoids. Once formed, organoids were replated at a 1:4 split ratio and treatment with 400 nM 4-OH TAM was initiated after 24 h to induce Cbl/Cblb deletion. (A) Bright-field imaging showed that while FF control mouse intestinal organoids exhibited a steady increase in budding (crypt domains) over time (up to 72 h of observation), the FF/creERT mouse organoids showed increased budding until 48 h after 4-OH-TAM induction but rapidly lost crypt domains by 72 h post-induction; scale bar = 40 μm. (B) Representative H&E-stained images of FF control and FF/creERT organoids confirm the loss of morphological features in the latter; scale bar = 50 μm. (C) Ten distinct organoids per genotype were followed up to 72 h and change in the number of buds (crypt domains) at each time point relative to time 0 was quantified. (D) FF Control and FF/creERT organoids grown in 4-OH-TAM for 72 h were re-passaged and imaged after 48 h. Note the lack of organoid structures with intact morphology in FF/creERT organoid cultures, supporting a loss of self-renewal contrary to growth and intact morphology of control organoids; scale bar = 400 μm. (E) Immunoblotting of organoid lysates at different time points confirmed effective Cbl/Cblb deletion by 48 and 72 h time points in FF/creERT organoids. HSC-70, loading control. FF/creERT organoids derived from two independent female mice (2F and 3F) were cultured in the presence (iDKO) or absence (control) of 400 nM 4-hydroxy-TAM for 72 h. Single cells isolated from the organoids were processed through the GemCode Single Cell Platform (10X Genomics) to perform single-cell RNA-seq and analyzed using Seurat. (F) tSNE map of combined controls and combined Cbl/Cblb iDKO mouse organoid cells. Cells are grouped into seven clusters based on transcriptome profiles and are colored accordingly. The cell types were assigned based on gene expression profile of stem cells ( Lgr5, Ascl2, Axin2, Olfm4, Gkn3 ), transit-amplifying (TA) cells ( Mki67 , Cdk4 , Mcm5 , Mcm6 , Pcna ), enterocytes ( Alpi , Apoa1 , Apoa4 , Fabp1 ), Paneth cells ( Lyz1 , Defa17 , Defa22 , Defa24 , Ang4 ), enteroendocrine cells ( Chga , Chgb , Tac1 , Tph1 , Neurog3 ), goblet cells ( Muc2 , Tff3 , Agr2 ) and tuft cells ( Dclk1 , Trpm5 , Gfi1b ). (G) Heatmap shows the expression levels of top cluster-specific genes in each cluster. Yellow represents the highest expression while purple represents low or no expression. (H) Histogram depicting the percentage of cells in each cluster. (I) Validation of Cbl and Cblb deletion in the 2F and 3F organoids by qPCR. (J) Gene set enrichment analysis (GSEA) of each cell type between control and iDKO organoids shows upregulation the of PI3K-Akt-mTOR signaling pathway upon Cbl/Cblb iDKO in stem cells, goblet cells, enteroendocrine cells, Paneth cells, and enterocytes. NES (normalized enrichment score), FDR (false discovery rate), and p values are indicated in the GSEA plots. (K) To validate the alterations in PI3K-mTOR signaling upon Cbl/Cblb iDKO, organoid lysates were collected at different time points of culture in the presence of 4-OH-TAM and analyzed by immunoblotting for EGFR, p -EGFR, Akt, p -Akt, S6 and p-S6. Clear upregulation of p -AKT/mTOR pathway components ( p -Akt and p-S6) is seen and pEGFR upregulation is observed only after 72 h of 4-OH-Tam induction. Hsc-70 served as a loading control. Densitometries of Cbl, Cbl-b, p -EGFR/EGFR, p -Akt/Akt and p-S6/S6 after normalizing to loading control in comparison to FF control sample in each time point are indicated on the top of the immunoblots. (L) Intestinal tissue sections from Control and Cbl/Cblb iDKO mice 10 days after initiating TAM treatment were stained with antibodies against p-S6 to assess the impact of Cbl/Cblb deletion. Sections stained with secondary antibody alone were used as a negative control. p-S6 levels (red in color) were sharply increased in iDKO as compared to control sections; scale bar = 20 μm. Quantified data are presented as mean ± SEM of three independent experiments with statistics using student’s two-tailed t test . ns, not significant; p ≤ 0.05, ∗; p ≤ 0.01, ∗∗; p ≤ 0.001, ∗∗∗.

Article Snippet: Rabbit Polyclonal anti-Phospho EGFR (Y1068) , Cell Signaling Technology , Cat#2234; RRID: AB_331701.

Techniques: Isolation, Control, Imaging, Staining, Western Blot, Derivative Assay, Cell Culture, RNA Sequencing, Gene Expression, Expressing, Biomarker Discovery, Comparison, Negative Control, Two Tailed Test

Journal: iScience

Article Title: Cbl and Cbl-b ubiquitin ligases are essential for intestinal epithelial stem cell maintenance

doi: 10.1016/j.isci.2024.109912

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal anti-Phospho EGFR (Y1068) , Cell Signaling Technology , Cat#2234; RRID: AB_331701.

Techniques: Recombinant, In Vivo, In Vitro, Plasmid Preparation, Staining, Polymer, Blocking Assay, Amplification, Isolation, Reverse Transcription, SYBR Green Assay, Western Blot, Bicinchoninic Acid Protein Assay, Knock-In, Control, Software, Flow Cytometry