primary antibodies against bmal1  (Cell Signaling Technology Inc)

Journal: eBioMedicine

Article Title: Circadian rhythm disruption impairs ovarian follicular development via NAD + metabolic reprogramming

doi: 10.1016/j.ebiom.2026.106200

Figure Lengend Snippet: Morphological and functional changes of ovaries between Ctrl and LP groups. (a) The illumination pattern for SD rats. Time (hours) refers to clock time. (b) Serum melatonin levels of Ctrl (n = 4) and LP (n = 4) group of every ZT. (c) The expression phase of Bmal1 in ovaries. (d) Typical oestrous cycles from Ctrl (n = 12) and LP (n = 12) group. Proestrus (P), oestrus (E), metoestrus (M), dioestrus (D). (e) Composition of oestrous cycle (n = 12). (f) Ovaries from Ctrl (n = 3) and LP (n = 3) group. (Scale bar, 1 cm). (g) Ovary weight of Ctrl (n = 9) and LP (n = 7) group. (h) HE staining images of ovary section for rats, triangle means expanded cystic follicles, pentagram means corpus luteum. Scale bar, 1 mm. (i) Comparison of follicle numbers of Ctrl (n = 4) and LP (n = 6) group. (j) BrdU immunohistochemical staining (Scale bar, 500 μm). (k) The number of retrieved oocytes (n = 5) after ovulation induction. (l) Litter size of Ctrl (n = 24) and LP (n = 11) groups. Statistical differences between the Ctrl and LP groups were determined using Tukey's test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Samples were incubated overnight at 4 °C with rotation using primary antibodies against BMAL1 (Cell Signalling Technology, 14020; 2 μg) or appropriate normal IgG (ABclonal, AS070; 2 μg), followed by incubation with secondary antibodies (Millipore, AP132; 1:100 dilution) for 1 h at room temperature.

Techniques: Functional Assay, Expressing, Staining, Comparison, Immunohistochemical staining

Journal: eBioMedicine

Article Title: Circadian rhythm disruption impairs ovarian follicular development via NAD + metabolic reprogramming

doi: 10.1016/j.ebiom.2026.106200

Figure Lengend Snippet: Disruption of NAMPT oscillated expression through NAMPT after long photoperiod exposure. (a) Ovaries were obtained at different ZT from Ctrl and LP group for WB, β-Tubulin was used as control. (b) The relative expression of BMAL1 at different ZT based on ZT0 to show the dynamic changes of Ctrl and LP group (n = 3). (c) The relative expression of NAMPT at different ZT based on ZT0. (d) WB of ovaries from Ctrl and LP group at each ZT (n = 3). (e) BMAL1 expression of LP group relative to Ctrl at each ZT (n = 3). (f) NAMPT expression of LP group relative to Ctrl at each ZT (n = 3). (g) Linear correlation of NAMPT and BMAL1 expression in Ctrl and LP group, which were showed for calculated expression based on its fold change to raw expression of ZT0. (h) Genomic views of BMAL1 CUT&Tag-seq assay enrichment at the promoters of the Nampt . (i) The non-canonical E-box motifs in the promoter and first intron of the Nampt gene. Chromatin immunoprecipitation (ChIP) qPCR was used to show that BMAL1 physically and specifically associate to the E-boxes on the Nampt promoter, since the comparative analysis with 1% input as a reference revealed a significant decrease in fold change at the LP group site. (j) BMAL1-NAMPT-SIRT3-Deacetylation SOD2 axis. Statistically significant differences between the Ctrl and LP groups were determined using Tukey's test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Samples were incubated overnight at 4 °C with rotation using primary antibodies against BMAL1 (Cell Signalling Technology, 14020; 2 μg) or appropriate normal IgG (ABclonal, AS070; 2 μg), followed by incubation with secondary antibodies (Millipore, AP132; 1:100 dilution) for 1 h at room temperature.

Techniques: Disruption, Expressing, Control, Chromatin Immunoprecipitation, ChIP-qPCR

Journal: Oral oncology

Article Title: The circadian clock gene, BMAL1, promotes radiosensitization in nasopharyngeal carcinoma by inhibiting the epithelial-to-mesenchymal transition via the TGF-β1/Smads/Snail1 axis.

doi: 10.1016/j.oraloncology.2024.106798

Figure Lengend Snippet: Fig. 1. The establishment, biobehavioral validation, and cell transfection of radiation-resistant cell line 5-8FR (A) The amount of BMAL1 protein expressed in nasopharyngeal cancer cells is inversely proportional to the radiation dose; (B) The cell clonal formation of two groups of cells at different irradiation doses; (C) Cell clonal formation rates of 5-8F and 5-8FR under different irradiation doses; (D) The cell survival fraction curves of 5-8F and 5-8FR at different irradiation doses. (E) The expression of EMT-associated proteins in 5-8F and 5-8FR, detected by Western blot; (F) The proliferation abilities of 5-8F and 5-8FR, detected by cell counting kit-8 assays; (G,I) The migration abilities of 5-8F and 5-8FR within 24 h, detected by wound-healing tests (×40); (H,J) The migration and invasion abilities of 5-8F and 5-8FR within 24 h, detected by Transwell assays (×200); (K) GFP green fluorescence was observed 48 h after transfection in 5-8FR under an inverted fluorescence microscope (×200); (L) Grayscale images of BMAL1 protein expression in 5-8FR after transfection, detected by WB. (*p < 0.05, **p < 0.01, ***p < 0.001, nsp > 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After performing antigen retrieval, nonspecific binding sites were blocked with 3 % BSA for 30 min. Primary antibodies against BMAL1 (1:2000; catalog number: 20847–1-AP; Proteintech) were applied to the sections and incubated at 4 ◦C overnight.

Techniques: Biomarker Discovery, Transfection, Irradiation, Expressing, Western Blot, Cell Counting, Migration, Fluorescence, Microscopy

Journal: Oral oncology

Article Title: The circadian clock gene, BMAL1, promotes radiosensitization in nasopharyngeal carcinoma by inhibiting the epithelial-to-mesenchymal transition via the TGF-β1/Smads/Snail1 axis.

doi: 10.1016/j.oraloncology.2024.106798

Figure Lengend Snippet: Fig. 2. Effect of the BMAL1 gene on the radiotherapy sensitivity, proliferation, migration, invasion capacity and EMT of the radiation-resistant cell line 5-8FR (A) The clonal formation of 5-8FR BMAL1-OE and control group cells at different irradiation doses; (B) Cell clonal formation rate of 5-8FR BMAL1-OE and control group cells under different irradiation doses; (C) The cell survival fraction curve of 5-8FR BMAL1-OE and control group cells at different irradiation doses; (D) WB was used to detect the effect of the BMAL1 gene on EMT-related protein expression in 5-8FR cells induced by recombinant human TGF-β1 (E) The cell counting kit-8 assay was used to detect the effect of recombinant human TGF-β1 induction on the proliferation capacity of 5-8FR cells; (F) The cell counting kit-8 assay was used to detect the effect of the BMAL1 gene on the proliferation capacity of 5-8FR cells under the induction of recombinant human TGF-β1; (G-H) The wound-healing test was used to detect the effect of the BMAL1 gene on the migration ability of 5-8FR cells under the induction of recombinant human TGF-β1(×40); (I-J) Transwell assay was used to detect the effect of the BMAL1 gene on the migration and invasion ability of 5-8FR cells under the induction of recombinant human TGF-β1 (×200). (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, nsp > 0.05).

Article Snippet: After performing antigen retrieval, nonspecific binding sites were blocked with 3 % BSA for 30 min. Primary antibodies against BMAL1 (1:2000; catalog number: 20847–1-AP; Proteintech) were applied to the sections and incubated at 4 ◦C overnight.

Techniques: Migration, Control, Irradiation, Expressing, Recombinant, Cell Counting, Transwell Assay

Journal: Oral oncology

Article Title: The circadian clock gene, BMAL1, promotes radiosensitization in nasopharyngeal carcinoma by inhibiting the epithelial-to-mesenchymal transition via the TGF-β1/Smads/Snail1 axis.

doi: 10.1016/j.oraloncology.2024.106798

Figure Lengend Snippet: Fig. 3. The BMAL1 gene improves radiosensitivity of NPC by inhibiting the TGF-β1/Smads/Snail1 axis to inactivate the EMT in vitro (A-C) The BMAL1 gene inhibits the expression of TGF-β1/Smads pathway-related proteins in 5-8FR; (D,E) Flow cytometry was used to detect the effect of the BMAL1 gene on the apoptosis rate of 5- 8FR cells after 10 Gy irradiation under the induction of recombinant human TGF-β1; (F,G) The EDU method was used to detect the effect of the BMAL1 gene on the DNA synthesis ability of 5-8FR cells 72 h after 10 Gy irradiation under the induction of recombinant human TGF-β1(×200); (H) Gray scale of p-Smad2 and Snail1 protein expression in the nuclei and cytoplasm of each group; (I) p-Smad2 protein expression in the cytoplasm of each group; (J) p-Smad2 and Snail1 protein expression in the nuclei of each group. (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: After performing antigen retrieval, nonspecific binding sites were blocked with 3 % BSA for 30 min. Primary antibodies against BMAL1 (1:2000; catalog number: 20847–1-AP; Proteintech) were applied to the sections and incubated at 4 ◦C overnight.

Techniques: In Vitro, Expressing, Flow Cytometry, Irradiation, Recombinant, DNA Synthesis

Journal: Oral oncology

Article Title: The circadian clock gene, BMAL1, promotes radiosensitization in nasopharyngeal carcinoma by inhibiting the epithelial-to-mesenchymal transition via the TGF-β1/Smads/Snail1 axis.

doi: 10.1016/j.oraloncology.2024.106798

Figure Lengend Snippet: Fig. 4. Induced by recombinant human TGF-β1, the BMAL1 gene inhibits EMT and its acquired radio-resistance in NPC (a: OENC+TGF-β1, b: OENC, c: OE+TGF-β1) (A) Nude mice were subjected to subcutaneous tumors and sacrificed after radiotherapy; (B) The volume growth curves of transplanted tumors in each group before radiotherapy; (C) The volume change curves of transplanted tumors in each group after radiotherapy; (D) After radiotherapy, the volume reduction ratio of transplanted tumors in each group was statistically significant; (E) Weight statistics of transplanted tumors in each group; (F,H) TUNEL-detected apoptosis in each group of transplanted tumors; (I) HE staining-detected pathological characteristics for each group of transplanted tumors (×100) and immunohistochemistry- detected expression changes in the E-cadherrin protein in transplanted tumor tissues (×100, ×200); (G) Immunohistochemical score of the E-cadherrin protein in nude mouse transplant tumors of NPC. (J) Mechanism simulation diagram of BMAL1 inhibiting EMT and promoting the radiosensitization of NPC through the TGF- β1/Smads/Snail1 axis. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: After performing antigen retrieval, nonspecific binding sites were blocked with 3 % BSA for 30 min. Primary antibodies against BMAL1 (1:2000; catalog number: 20847–1-AP; Proteintech) were applied to the sections and incubated at 4 ◦C overnight.

Techniques: Recombinant, TUNEL Assay, Staining, Immunohistochemistry, Expressing, Immunohistochemical staining

Journal: European Thyroid Journal

Article Title: Circadian clock disruption in autoimmune thyroiditis

doi: 10.1530/ETJ-23-0035

Figure Lengend Snippet: Altered core clock gene expression in patients with AIT. (A) Comparisons of relative mRNA levels of core clock genes in thyroid tissues of patients with AIT and control ( n = 25–30/group). (B) Representative IHC sections of BMAL1 and PER2 for thyroid samples from the same AIT and control subject (counterstained with Hematoxylin), yellow arrow indicates the positive staining area (magnification = ×400; scale bar = 50 μm). (C) Quantification of BMAL1 and PER2 by IHC intensity ( n = 30 per group). Data represent the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant; two-way significance was calculated by Mann–Whitney U test.

Article Snippet: Primary antibodies against BMAL1 (NB100-2288, dilution 1:500; Novus, USA) and PER2 (NB100-125, dilution 1:250; Novus) and HRP-conjugated secondary antibodies (1:2000) supplied in a DAB kit (CW2069S, CWBIO, Beijing, China) were used for immunostaining of thyroid sections.

Techniques: Gene Expression, Control, Staining, MANN-WHITNEY

Journal: European Thyroid Journal

Article Title: Circadian clock disruption in autoimmune thyroiditis

doi: 10.1530/ETJ-23-0035

Figure Lengend Snippet: Association between inflammatory biomarker and mRNA levels of clock genes. (A) Elevated serum inflammatory cytokines in AIT patients compared with control group. (B) Increased mRNA expression of pro-inflammatory genes in thyroid samples from AIT patients compared with control group. n = 25–30/group (C) Associations of BMAL1 and PER2 with inflammatory biomarkers, multivariate linear regression models were adjusted for age, sex, BMI, and time of sample collection. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant; two-way significance was calculated by Mann–Whitney U test.

Article Snippet: Primary antibodies against BMAL1 (NB100-2288, dilution 1:500; Novus, USA) and PER2 (NB100-125, dilution 1:250; Novus) and HRP-conjugated secondary antibodies (1:2000) supplied in a DAB kit (CW2069S, CWBIO, Beijing, China) were used for immunostaining of thyroid sections.

Techniques: Biomarker Discovery, Control, Expressing, MANN-WHITNEY