co ip experiment  (Cell Signaling Technology Inc)

Journal: Cell Death Discovery

Article Title: The RNF8/OPTN/KDM6A axis controls macrophage polarization to maintain testicular microenvironment homeostasis

doi: 10.1038/s41420-025-02641-3

Figure Lengend Snippet: A , B Detected and statistically analyzed the expression levels of KDM6A in sh-RNF8 293T cells with successful transduction (white arrow) and unsuccessful transduction (blue arrow), n = 50. Scale bar 20 µm. C , D And KDM6A expression levels were detected and statistically analyzed in oe-RNF8 293T cells with successful transduction (white arrow) and unsuccessful transduction (blue arrow), n = 57. Scale bar 20 µm. E Co-IP detection of KDM6A and RNF8 in 293T cells. F Binding response (nm) between KDM6A and RNF8 was measured by Octet R2 system. G 10 μM MG132 and 1 mM NH 4 Cl were treated with control 293T cells or oe-RNF8 293T cells for 6 h, respectively, to detect the protein degradation of KDM6A by western blot. H EBSS or 100 nM Rapamycin was used to treat 293T cells in the control group or knocked down the RNF8 group for 4 h, respectively, and the expression of KDM6A was detected by western blot. I , J 293T cells overexpressed or knocked down RNF8, IP-KDM6A, and detected the level changes of LC3I and LC3II enriched by KDM6A. K , L Knocked down or overexpressed RNF8, IP-KDM6A, and detected the ubiquitin, K48-linked ubiquitination, and K63-linked ubiquitination level enriched by KDM6A. All values were presented as the mean ± SD. Student’s t -test; * P < 0.05, ** P < 0.01, **** P < 0.0001. WB strips in this figure correspond to Fig. 6 in WB's original data.

Article Snippet: In the IP ubiquitin experiment, additional proteasome inhibitors (MG132, HY-13259, MCE; Epoxomicin, HY-13821, MCE) and deubiquitinase inhibitors (PR-619, HY-13814, MCE) need to be added.

Techniques: Expressing, Transduction, Co-Immunoprecipitation Assay, Binding Assay, Control, Western Blot, Ubiquitin Proteomics

Journal: Cell Death Discovery

Article Title: The RNF8/OPTN/KDM6A axis controls macrophage polarization to maintain testicular microenvironment homeostasis

doi: 10.1038/s41420-025-02641-3

Figure Lengend Snippet: A RNF8 ubiquitination modification omics data. B IP-RNF8 in 293T cells, silver stained. Exogenous ( C ) and endogenous ( D ) co-IP detection of RNF8 and OPTN. E , F Binding response (nm) between RNF8 and OPTN was measured by Octet R2 system. G The interaction between OPTN and SQSTM1 (p62) in the STRING database. H Endogenous co-IP results of OPTN and p62. I After treatment with MG132 or NH 4 Cl, the expression changes of OPTN, p62, and LC3 were detected. J HA-OPTN was transfected into 293T cells with knockdown or overexpression of RNF8, and its ubiquitin level was detected by IP-HA. K Transfected His-ub in 293T cells with knockdown or overexpression of RNF8, and detected the change of ubiquitin level of OPTN. L Transfected His-K63 in 293T cells with knockdown or overexpression of RNF8, and detected the change of K63 ubiquitin level of OPTN. M Transfected wild-type OPTN or K448R into 293T cells and detected the change of ubiquitin levels of OPTN. N After 48 h of starvation treatment in WT or KO mice, the changes in ubiquitin levels of OPTN in testicular tissue were detected. WB strips in this figure correspond to Fig. 7 in WB's original data.

Article Snippet: In the IP ubiquitin experiment, additional proteasome inhibitors (MG132, HY-13259, MCE; Epoxomicin, HY-13821, MCE) and deubiquitinase inhibitors (PR-619, HY-13814, MCE) need to be added.

Techniques: Ubiquitin Proteomics, Modification, Staining, Co-Immunoprecipitation Assay, Binding Assay, Expressing, Transfection, Knockdown, Over Expression

Journal: Clinical and Translational Medicine

Article Title: Oestrogen suppresses the adipogenesis of fibro/adipogenic progenitors through reactivating the METTL3–ESR1‐mediated loop in post‐menopausal females

doi: 10.1002/ctm2.70206

Figure Lengend Snippet: Oestrogen receptor 1 (ESR1) suppressed adipogenic differentiation of fibro/adipogenic progenitors (FAPs) by inhibiting transcription of peroxisome proliferator‐activated receptor gamma (PPARγ). (A) Bubble chart of GO analysis of up‐regulated and down‐regulated genes in FAPs from Esr1 knockout (KO) mouse when compared with those from Esr1 f/f mouse. Key GO terms were highlighted with red frames. (B) KEGG analysis of up‐regulated genes in FAPs from Esr1 KO mouse compared to those from Esr1 f/f mice. Key KEGG terms were highlighted with a red frame. (C) The sequence logo of potential ESR1 binding sites on the PPARγ promoter was predicted using JASPAR. (D) Scheme of construction of wild type (WT) and mutant pGL3‐PPARγ promoter reporter plasmids. (E, F) Protein levels and quantitative assessment of ESR1 and GAPDH after transfection with ESR1 overexpression (ESR1 OE) and vector (Vector) plasmids ( n = 3 per condition). (G) Quantitative assessment of luciferase activity after transfection with WT and mutant (Mut) pGL3‐PPARγ promoter reporter plasmids in female FAPs transfected with ESR1 overexpression plasmid ( n = 3 per condition). (H) Chromatin immunoprecipitation (ChIP)‐qPCR analysis for ESR1 binding to the PPARγ promoter in female FAPs under incubation with IgG or anti‐ESR1 antibodies ( n = 3 per condition). Data were shown as mean ± standard deviation (SD), ns indicated no significant differences, * indicated p < .05, *** indicated p < .001, **** indicated p < .0001.

Article Snippet: The chromatin immunoprecipitation (ChIP) experiment was conducted with the SimpleChIP Plus Kit (CST, cat#9005S).

Techniques: Knock-Out, Sequencing, Binding Assay, Mutagenesis, Transfection, Over Expression, Plasmid Preparation, Luciferase, Activity Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Incubation, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: Oestrogen suppresses the adipogenesis of fibro/adipogenic progenitors through reactivating the METTL3–ESR1‐mediated loop in post‐menopausal females

doi: 10.1002/ctm2.70206

Figure Lengend Snippet: Oestrogen receptor 1 (ESR1) enhanced expression of methyltransferase‐like 3 (METTL3) in turn by serving as a transcription factor in fibro/adipogenic progenitors (FAPs). (A, B) Dot blot and quantitative assessment of relative N6‐methyladenosine (m6A) methylation levels in FAPs from peri‐menopausal (Peri) and post‐menopausal patients (Post; n = 3 patients/group). (C, D) Dot blot and quantitative assessment of relative m6A methylation levels in FAPs from sham mouse (Control) and FAPs from ovariectomy (OVX) mouse ( n = 3 mice/group). (E) The sequence logo of potential ESR1 binding sites on the METTL3 promoter was predicted using JASPAR. (F) Scheme of wild type (WT) and mutant (Mut) pGL3‐METTL3 promoter reporter plasmids. (G, H) Protein levels and quantitative assessment of ESR1 and GAPDH in female FAPs transfected with vector (Vector) and ESR1 overexpression (ESR1 OE) plasmids ( n = 3 per condition). (I) Relative luciferase activity after transfection of WT and mutant (Mut) pGL3‐METTL3 promoter reporter plasmids in female FAPs transfected with ESR1 overexpression plasmid ( n = 3 per condition). (J) Chromatin immunoprecipitation (ChIP)‐qPCR for ESR1 binding to the METTL3 promoter in female FAPs under incubation with IgG or anti‐ESR1 antibodies ( n = 3 per condition). Data were shown as mean ± standard deviation (SD), ns indicated no significant differences, * indicated p < .05, *** indicated p < .001.

Article Snippet: The chromatin immunoprecipitation (ChIP) experiment was conducted with the SimpleChIP Plus Kit (CST, cat#9005S).

Techniques: Expressing, Dot Blot, Methylation, Control, Sequencing, Binding Assay, Mutagenesis, Transfection, Plasmid Preparation, Over Expression, Luciferase, Activity Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Incubation, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: Oestrogen suppresses the adipogenesis of fibro/adipogenic progenitors through reactivating the METTL3–ESR1‐mediated loop in post‐menopausal females

doi: 10.1002/ctm2.70206

Figure Lengend Snippet: E2 reactivated methyltransferase‐like 3 (METTL3)–oestrogen receptor 1 (ESR1)‐mediated loop in female post‐menopausal fibro/adipogenic progenitors (FAPs). (A) Chromatin immunoprecipitation (ChIP)‐qPCR for ESR1 binding to the peroxisome proliferator‐activated receptor gamma (PPARγ) promoter in female FAPs incubated with IgG or anti‐ESR1 antibodies following 3‐day treatments with either DMSO (Control) or oestrogen (E2) ( n = 3 per condition). (B) ChIP‐qPCR for ESR1 binding to the METTL3 promoter in female FAPs incubated with IgG or anti‐ESR1 antibodies following 3‐day treatments with either DMSO (Control) or oestrogen (E2) ( n = 3 per condition). (C, D) The protein levels of ESR1 and METTL3 in female post‐menopausal FAPs treated with DMSO (Control) or oestrogen (E2) ( n = 3 per condition). (E, F) Oil Red O staining and quantitative assessment of female post‐menopausal FAPs treated with DMSO (Control) or oestrogen (E2) following 10 days of adipogenic differentiation ( n = 5 per condition). Red indicated Oil Red O, blue indicated DAPI and the merged images were shown. Scale bar, 50 µm. (g) The mRNA expression of adipogenic and lipogenic genes in female post‐menopausal FAPs treated with DMSO (Control) or oestrogen (E2) following 10 days of adipogenic differentiation ( n = 3 per condition). Data were shown as mean ± standard deviation (SD), * indicated p < .05, ** indicated p < .01, *** indicated p < .001, **** indicated p < .0001.

Article Snippet: The chromatin immunoprecipitation (ChIP) experiment was conducted with the SimpleChIP Plus Kit (CST, cat#9005S).

Techniques: Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Incubation, Control, Staining, Expressing, Standard Deviation