Journal: bioRxiv

Article Title: MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription

doi: 10.1101/2024.06.30.601446

Figure Lengend Snippet: (A) Endogenous MeCP2 interacts with endogenous SEC components (AFF4, AF9, ENL, and ELL2) and RNA pol II in HEK293T cells. Normal mouse IgG was used as a negative control. (B) Endogenous MeCP2 interacts with SEC components (AFF4 and ELL2) and RNA pol II in the cortex of WT mouse at 7-weeks of age. (C) Reverse IP of endogenous AFF4 from cortical lysates of Camk2a-Cre/+ control (Cre) and Camk2a-Cre> Aff4 fl/fl ( Aff4 cKO) mice at 7-weeks of age. Mice were injected with either saline (baseline) or kainic acid (KA).

Article Snippet: 2μg of spike-in antibody (Active Motif; 61686) was added to each sample with one of the following antibodies for immunoprecipitations: 5μg of AFF4 antibody (Bosterbio; M03824) and 5μl of RNA pol II antibody (Cell Signaling Technology; 14958S).

Techniques: Negative Control, Control, Injection, Saline

Journal: bioRxiv

Article Title: MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription

doi: 10.1101/2024.06.30.601446

Figure Lengend Snippet: (A) shRNA-mediated knockdown of AFF4 in HEK293T cells abolishes the interaction between MeCP2 and the remaining components of the SEC (ENL and ELL2). In contrast, knockdown of ENL or ELL2 does not affect the interaction between MeCP2 and the remaining components of the SEC. NT sh is a non-targeting short-hairpin negative control. (B) In vitro binding assay between recombinant FLAG-tagged MeCP2 and HA-tagged SEC components. (C) In vitro binding assay between FLAG-tagged MeCP2 fragments and HA-tagged AFF4. The domains critical for interaction are highlighted in bold. Diagram on the right shows a map of the MeCP2 fragments that were tested. NTD = N-terminal domain; MBD = methyl-CpG-binding domain; ID = intervening domain; TRD = transcriptional repression domain; CTD = C-terminal domain. (D) In vitro binding assay between AFF4 and MeCP2 with deletions in the NTD, ID, TRD, ID and TRD, and CTD. The domains critical for interaction are in bold.

Article Snippet: 2μg of spike-in antibody (Active Motif; 61686) was added to each sample with one of the following antibodies for immunoprecipitations: 5μg of AFF4 antibody (Bosterbio; M03824) and 5μl of RNA pol II antibody (Cell Signaling Technology; 14958S).

Techniques: shRNA, Knockdown, Negative Control, In Vitro, Binding Assay, Recombinant

Journal: bioRxiv

Article Title: MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription

doi: 10.1101/2024.06.30.601446

Figure Lengend Snippet: (A) Heatmap of log occupancy of AFF4 in the cortex of WT and Mecp2 null mice. (B) Heatmap of log fold change of AFF4 occupancy in Mecp2 null mouse compared to WT mouse. (C) Heatmap of log occupancy of RNA pol II in the cortex of WT and Mecp2 null mice. (D) Heatmap of log fold change of RNA pol II occupancy in Mecp2 null mouse compared to WT mouse. 8696 RNA pol II-bound genes are represented in all heatmaps.

Article Snippet: 2μg of spike-in antibody (Active Motif; 61686) was added to each sample with one of the following antibodies for immunoprecipitations: 5μg of AFF4 antibody (Bosterbio; M03824) and 5μl of RNA pol II antibody (Cell Signaling Technology; 14958S).

Techniques:

Journal: bioRxiv

Article Title: MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription

doi: 10.1101/2024.06.30.601446

Figure Lengend Snippet: (A) Heatmap showing the log fold change of AFF4 and RNA pol II binding in the Mecp2 null cortex based on hierarchical clustering. (B) Two-dimensional plot showing the change in AFF4 vs. RNA pol II binding in Mecp2 null animals across each cluster. Color scale indicates the gene count. Data shown is the average of three biological replicates. Spearman’s correlation values: cluster I π=0.14, p=1.5e-10; cluster II π=0.09, p=8.2e-10; cluster III π=0.09, p=0.1. (C) Violin plot showing the average expression value of all genes in each cluster relative to WT cluster I. Data shown is the average of six biological replicates. Statistical significance was assessed using the Wilcoxon Rank Sum Test. (D) Gene ontology analysis of cluster III genes. All terms are FDR < 0.05. (E) Track examples of AFF4 and RNA pol II binding and RNA expression levels in WT (grey) and Mecp2 null (orange) mouse.

Article Snippet: 2μg of spike-in antibody (Active Motif; 61686) was added to each sample with one of the following antibodies for immunoprecipitations: 5μg of AFF4 antibody (Bosterbio; M03824) and 5μl of RNA pol II antibody (Cell Signaling Technology; 14958S).

Techniques: Binding Assay, Expressing, RNA Expression

Journal: Frontiers in Pharmacology

Article Title: Formononetin Improves the Survival of Random Skin Flaps Through PI3K/Akt-Mediated Nrf2 Antioxidant Defense System

doi: 10.3389/fphar.2022.901498

Figure Lengend Snippet: (A) The protein expression of HO-1, NQO1, GCLc, GCLm, TrxR1 and Keap1 was detected by western blot (B) The expressing levels of proteins relate to antioxidant/phase II detoxification enzymes. All data represent mean ± SD. * p ≤ 0.05 and ** p ≤ 0.01 compared to the control group.

Article Snippet: Primary antibodies against NQO1, TrxR1, HO-1, Keap1 IL-1β, TNF-α and Lamin B were from Abcam (Cambridge, UK); antibodies against GCLc and GCLm were purchased from Boster Biological Technology (Wuhan, China); antibodies against Nrf2, GAPDH, PI3K, P-Pi3k, AKT, and P-AKT were purchased from Cell Signaling Technologies (Danvers, MA, United States); antibodies directed vascular endothelial growth factor (VEGF) and SOD2 were obtained from Affinity Biosciences (Cincinnati, OH, United States).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Formononetin Improves the Survival of Random Skin Flaps Through PI3K/Akt-Mediated Nrf2 Antioxidant Defense System

doi: 10.3389/fphar.2022.901498

Figure Lengend Snippet: (A,B) Western blot along with its quantification revealed that the level of P-PI3K, P-Akt and Nrf2 as treated above (C,D) Western blot along with its quantification revealed that the level of HO-1, NQO1, GCLc, GCLm and TrxR1 (E,F) The IHC result of SOD2 and HO-1 expression in skin flaps (scale bar: 50 μm) ** p ≤ 0.01 compared to the control group; ## p ≤ 0.01 compared to the FMNT-H group.

Article Snippet: Primary antibodies against NQO1, TrxR1, HO-1, Keap1 IL-1β, TNF-α and Lamin B were from Abcam (Cambridge, UK); antibodies against GCLc and GCLm were purchased from Boster Biological Technology (Wuhan, China); antibodies against Nrf2, GAPDH, PI3K, P-Pi3k, AKT, and P-AKT were purchased from Cell Signaling Technologies (Danvers, MA, United States); antibodies directed vascular endothelial growth factor (VEGF) and SOD2 were obtained from Affinity Biosciences (Cincinnati, OH, United States).

Techniques: Western Blot, Expressing, Control

Journal: Redox Biology

Article Title: Competition of nuclear factor-erythroid 2 factors related transcription factor isoforms, Nrf1 and Nrf2, in antioxidant enzyme induction ☆

doi: 10.1016/j.redox.2013.01.005

Figure Lengend Snippet: Age-dependent alteration in protein binding to the GCLM EpRE. Protein extracts from lung samples of young (6-month-old) and middle-aged (21-month-old) mice were analyzed for their ability to bind to a biotinylated EpRE from human GCLM promoter with EMSA (gel-shift) assay as described in . (A) Age-increased EpRE binding. Representative blot and band densitometry results ( N =5);⁎, p -value<0.05, (Rank sum test). (B) Immunodepletion experiments to determine relative contribution of Nrf1, Nrf2, and Bach1 to the probe. Percentage of immunodepletion (compared to IgG) is shown ( N =5); ⁎, p -value=0.056, n.s., not significantly different (Rank sum test).

Article Snippet: The EMSA kit, including biotinylated EpRE probe from the human glutamate–cysteine ligase, modifier subunit ( gclm ) promoter was purchased from (Panomics, Fremont, CA).

Techniques: Protein Binding, Gel Shift, Binding Assay, Immunodepletion