Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) The left graph shows the X-ray crystal structure of a p50 / p65 heterodimer bound to DNA as published in (PDB 1kvx), while the right graph shows the entire p65 protein structure including the disordered C-terminal half as calculated by alphafold ( https://alphafold.ebi.ac.uk/entry/Q04206 ). Residues required for dimerization (Phe (F) 213, Leu (L) 215) or DNA binding (Glu (E) 39) are indicated in both structures. (B) Scheme of the HA-tagged p65-miniTurbo fusion proteins that were used to reconstitute p65-deficient HeLa cells under the control of a tetracycline-sensitive promoter. F213 and L215 in p65 wildtype (wt) were mutated to Asp (FL / DD) for dimerization-deficient p65 or E39 to Ile (E / I) for DNA-binding-deficient p65. (C) Principle of proximity-based biotin tagging. (D) Pools of HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected (using branched Polyethyleneimine, PEI)) with the constructs shown in (B) and their expression was induced with doxycycline (1 µg / ml) for 17 h. At the end of this incubation, intracellular biotinylation was induced by adding 50 µM biotin for 70 minutes as indicated. Additionally, half of the samples were treated with IL-1α (10 ng / ml) for the last 60 minutes. Cell cultures expressing HA-miniTurbo only (empty vector, EV) or receiving only doxycycline or biotin served as negative controls (indicated by gray font). Parental HeLa cells (p) were included as further controls. Left panel: Cells were lysed and proteins were analyzed by Western blotting for the expression of p65-HA-miniTurbo and HA-miniTurbo using anti p65 and anti HA antibodies. Equal loading was confirmed by probing the blots with anti β-actin antibodies. Right panel: Biotinylated proteins from the same samples were purified on streptavidin agarose beads and biotinylation patterns were visualized by Western blotting using streptavidin-horseradish peroxidase (HRP) conjugates (representative images from two independent experiments). (E) Biotinylated proteins from the experiment shown in (C) and from a second biological replicate were identified by mass spectrometry. Volcano plots show the ratio distributions of Log 2 -transformed mean protein intensity values on the X-axes obtained with wild type p65 or the p65 mutants compared to the empty vector controls in the presence or absence of IL-1α treatment. Y axes show corresponding p values from t-test results. Strong enrichment of the bait p65 / RELA proteins together with the core canonical NF-kB components is shown in red and blue colors, respectively (two biologically independent experiments and three technical replicates per sample). (F) Specific proteins binding to p65 / RELA wild type were defined by significant enrichment (LFC ≥ 2, -log 10 p ≥ 1.3) compared to HA-miniTurbo only and to cells exposed to doxycycline or biotin only (see ). This set of proteins was intersected with proteins enriched in cells expressing p65 mutant proteins (LFC ≥ 2, -log10 p ≥ 1.3). Venn diagrams show the numbers of p65 / RELA interactors and their overlaps before and after IL-1α-treatment, with values in the lower left corners indicating total numbers of interactors. (G) The six protein sets shown in (E) were subjected to parallel overrepresentation pathway analysis using Metascape software . The Venn diagrams show the overlap of the top 100 enriched pathway terms. For IL-1α samples, only 92 terms were enriched. Values in the lower left corners indicate total numbers of unique pathways. (H) The table shows the most strongly enriched pathway categories associated with the p65 / RELA wild type or mutant interactomes. Numbers in brackets indicate the total numbers of p65 / RELA interactors per condition that were subjected to overrepresentation analysis according to (E, F). The mass spectrometry data and bioinformatics analysis results are provided in Supplementary Table 1. See also and . rtTA, reverse tetracycline-controlled transactivator.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Binding Assay, Control, CRISPR, Transfection, Construct, Expressing, Incubation, Plasmid Preparation, Western Blot, Purification, Mass Spectrometry, Transformation Assay, Mutagenesis, Software

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Parental HeLa cells or pools of HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected with empty vector (EV) encoding HA-miniTurbo (HA-mTb) or with p65 / RELA wild type (wt) fused C-terminally to HA-mTb (p65(wt)-HA-mTb) as described in the legend of . The expression of the constructs was induced with increasing concentrations of doxycycline for 17 h as indicated. At the end of the incubation, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Cell extracts were analyzed by Western blotting for the expression of the p65-HA-mTb fusion protein or HA-mTb using polyclonal antibodies raised against the C-terminus of p65 / RELA (sc-372) or a monoclonal antibody raised against N-terminal amino acids 1-286 of p65 / RELA (sc-8008), or an anti HA antibody, respectively. Note that the fusion protein is better recognized with the N-terminal antibody preparations. (B) HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected with the indicated constructs and their expression was induced with doxycycline at 1 µg / ml for 17 h. On the next day, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Total RNA was isolated and analyzed by RT-qPCR for expression of the indicated genes. Bar graphs show means ± s.d. from two biologically independent experiments. (C) Cells were transfected as in (A) and expression of the p65 / RELA fusion protein was induced 20 h later with doxycycline (10 ng / ml) for 4 h. In last period of this incubation, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Cells were lysed and cytosolic (C), soluble nuclear (N1) and insoluble, chromatin nuclear fractions (N2) were analyzed by Western blotting for the expression and distribution of p65(wt)-HA-mTb. Antibodies against RNA polymerase II, tubulin and β-actin were used to control purity of fractions and equal loading.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: CRISPR, Transfection, Plasmid Preparation, Expressing, Construct, Incubation, Western Blot, Isolation, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Biotinylated proteins from the experiments shown in and from a second biological replicate were identified by mass spectrometry in the presence or absence of IL-1α treatment of cells. Volcano plots show the ratio distributions of Log 2 transformed mean protein intensity values obtained with wild type p65 in the presence of doxycycline and biotin (wt) compared to the empty vector control (EV) or compared with conditions in which only biotin (wt(bio)) or doxycycline (wt(dox)) were added to the cell cultures, to determine false positive values in the absence of expression of fusion protein but facilitated biotinylation, or in the absence of biotinylation but induced expression of the fusion protein, respectively. X-axes show mean ratio value and Y-axes show p values from t-test results. Strong enrichment of the bait p65 / RELA proteins together with the core canonical NF-kB components is shown in red and blue colors, respectively (two biologically independent experiments and three technical replicates per sample). (B) Specific proteins binding to p65 / RELA wild type were defined by significant enrichment (LFC ≥ 2, -log 10 p ≥ 1.3) compared to HA-miniTurbo only and to cells exposed to doxycycline or biotin only as shown in (A). Venn diagrams show the total numbers of specific p65 / RELA interactors and their overlaps before and after IL-1α-treatment. The intersecting 279 (without IL-1α) and 310 (with IL-1α) interactors were pooled, resulting in the set of 366 specific p65 / RELA interactors that was used for further downstream analyses. Numbers in the left lower corner of the boxes indicate the total number of detected interactors.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Mass Spectrometry, Transformation Assay, Plasmid Preparation, Control, Expressing, Binding Assay

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Protein interaction network of the 46 known p65 / RELA interactors found by miniTurboID. Edge widths visualize the evidence for experimental interactions deposited in the STRING database . Nodes are colored in red and are arranged according to the enrichment found by proximity labeling in our study. (B) Venn diagram of p65 / RELA interactors in IL-1α or untreated cells revealing a total of 366 unique p65 / RELA interactors, of which 320 (87.4 %) have no documented protein interaction entries in STRING. (C) Overlap of the RELA interactome with 1639 human TFs and 801 epigenetic regulators . (D) Graphs visualizing the top 10 enriched epigenetic regulators. Volcano plots show the ratio distributions of Log 2 transformed mean protein intensity values obtained with wild type p65 / RELA (wt) or with p65 / RELA mutants (FL/DD, E/I) compared to empty vector controls (EV). Only 9 reader proteins were found. (E) Association of enriched epigenetic regulators with known epigenetic complexes according to the annotation provided by . Numbers in brackets show identified components per complex. (F) Venn diagram showing the overlap of enriched TFs in basal or IL-1α-stimulated conditions. (G) Volcano plots visualizing all TFs significantly enriched with wt p65 / RELA (LFC ≥ 2, -log 10 p ≥ 1.3) compared with empty vector control (EV) and the changes obtained with p65 mutants in basal conditions. (H) Distribution of TF families found to be associated with p65 / RELA in basal and IL-1α-stimulated conditions according to the annotation provided by (I) IL-1α-dependent enrichment of all TF belonging to ZBTB and ZNF families as identified by miniTurboID. (J) The top 10 pathway terms according to GO (BP, CC, MF), KEGG, Reactome, STRING clusters and WikiPathways data base entries and the top 10 subcellular localizations associated with the 366 p65 / RELA interactors. Annotations, number of components and false discovery rates (FDR) were retrieved using the STRING plugin of Cytoscape . The mass spectrometry data sets and bioinformatics analysis results are provided in Supplementary Table 1.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Labeling, Transformation Assay, Plasmid Preparation, Control, Mass Spectrometry

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Final list of top ranking high confidence interactors p65 / RELA selected for further studies. The heatmap shows the Log 2 transformed mean protein intensity values from technical triplicates of the two biological independent miniTurboID experiments, the enrichment ratio values compared to the empty vector (HA-miniTurbo) control (EV) and the regulation by IL-1α. With the exception of N4BP3, all proteins were identified by at least two peptides. (B) Graph showing that the top 38 p65 / RELA interactors are largely devoid of known protein interactions based on STRING entries. According to STRING, only two factors (CEBPD and FOSL1) interact with p65 /RELA. Node borders visualize the main functional annotations. (C) HeLa cells were transiently transfected for 48 h with 20 nM of siRNAs mixtures for 38 HCI and p65 / RELA, a siRNA targeting luciferase, transfection reagent alone or were left untreated (untr.). Half of the cells per plate were treated for 1 h with IL-1α (10 ng / ml) at the end of the incubation. cDNAs were transcribed in lysates and amplicons for three NF-kB target genes, two housekeeping genes and all 38 HCI p65 / RELA interactors were pre-amplified by linear PCR and then quantified by qPCR. Based on Ct values, mRNA levels were quantified and normalized against GUSB . The effects of knockdowns were calculated separately for basal and IL-1α-inducible conditions against the luciferase siRNA. The heatmap shows hierarchically Kmeans clustered mean ratio values derived from three biologically independent siRNA screens. As a positive control, RELA knockdowns were performed in parallel. Green colors highlight p65 / RELA interactors selected for further analysis. (D) The miniTurboID enrichment of six p65 / RELA interactors (green colors) chosen from (C) is shown. The complete set of data of the screen is provided in Supplementary Table 2. See also .

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Transformation Assay, Plasmid Preparation, Control, Functional Assay, Transfection, Luciferase, Incubation, Amplification, Derivative Assay, Positive Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Scheme illustrating the arrangement of siRNAs and controls on individual cell culture plates and the performance of RT-qPCR measurements in cell extracts without prior RNA purification. A linear PCR amplification step was included to pre-amplify specific transcripts. (B) Confirmation of knockdown of 38 HCI and of RELA mRNAs by RT-qPCR as shown in (A). Bar graphs show mean changes ± s.d. relative to the luciferase siRNA controls (siLuci) from three biologically independent experiments.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Cell Culture, Quantitative RT-PCR, Purification, Amplification, Knockdown, Luciferase

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: Proximity-ligation assays coupled to immunofluorescence (IF) were performed with HeLa cells or Δp65 HeLa cells lacking endogenous p65 / RELA to demonstrate interactions of p65 / RELA with TFE3 (A), TFEB (B), GLIS2 (C) and ZBTB5 (D) using pairs of antibodies as indicated. PLA-spots are colored in red, while p65 IF is colored in green. Nuclear DNA is counterstained with Hoechst (blue signals). The images show representative fluorescence raw data and the violin plots on the right show quantification from the numbers of cells indicated in brackets. Samples omitting one of the two antibodies or both primary antibodies (ctr) served as negative controls. Solid lines indicate medians and dashed lines indicate 1 st and 3 rd quartiles. Asterisks indicate results from Kruskal-Wallis tests compared to the parental control (****p ≤ 0.0001). obtained by one-way ANOVA.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Ligation, Immunofluorescence, Fluorescence, Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to analyze the influences of novel p65 / RELA interactors on basal p65 / RELA target genes by combining siRNA-mediated knockdown with transcriptome analysis. (B) HeLa cells were transiently transfected for 48 hours with 20 nM siRNA mixtures against RELA, ZBTB5, S100A8, S100A9 (series 1) or RELA, GLIS2, TFE3, TFEB (series 2) and an siRNA against luciferase (siLuc) as control. Half of the cells were treated with IL-1α (10 ng/ml) for 1 hour at the end of incubation, and Agilent microarray analyses were performed from total RNA. Normalized data were used to identify DEGs based on an LFC ≥ 1 with a -log 10 p value ≥ 1.3. Venn diagrams show the overlap of all DEGs that were affected at least twofold by siRNA knockdown in untreated, basal conditions, with the ratio of siLuc to individual knockdown determined in each case. Red colors mark genes jointly regulated by knockdown of RELA and one of its interactors (two biologically independent experiments). (C) Violin plots show the distribution, medians, and interquartile ranges of normalized expression levels for all constitutively expressed genes and the corresponding changes in the gene subsets defined in that were affected by siRNA knockdown. The number of these genes is indicated in parentheses. (D) Superimposed pairwise correlation analyses of the mean ratio changes of all genes (gray), and gene sets significantly up- or down-regulated by siRNA knockdown (red). Ratio values from RELA knockdown conditions were compared with the knockdown of a RELA interactor in each case. Genes that are jointly regulated by knockdown of RELA and one of its interactors correspond to the Venn diagrams of (B) and are marked in red. Coefficients of correlation (Pearson’s r), corresponding p values and coefficients of determination (r 2 ) rare indicated for all comparisons. The complete set of data is provided in Supplementary Table 3.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Knockdown, Transfection, Luciferase, Control, Incubation, Microarray, Expressing

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to analyze the influences of novel p65 / RELA interactors on IL-1α-regulated p65 / RELA target genes by combining siRNA-mediated knockdown with transcriptome analysis. (B) HeLa cells were transiently transfected for 48 h with 20 nM siRNA mixtures against RELA, ZBTB5, S100A8, S100A9 (series 1) or RELA, GLIS2, TFE3, TFEB (series 2) and an siRNA against luciferase (siLuc) as control. Half of the cells were treated with IL-1α (10 ng/ml) for 1 hour at the end of incubation, and Agilent microarray analyses were performed from total RNA. Normalized data were used to identify DEGs based on an LFC ≥ 1 with a -log 10 p value ≥ 1.3. Venn diagrams show the overlap of all DEGs that were affected at least twofold by siRNA knockdown in IL-1α-treated samples, with the ratio of siLuc to individual knockdown determined in each case. Red colors mark genes jointly regulated by knockdown of RELA and one of its interactors (two biologically independent experiments). (C) Violin plots show the distribution, medians, and interquartile ranges of normalized expression levels for all IL-1α-regulated genes and the corresponding changes in the gene subsets defined in that were affected by siRNA knockdown. The number of these genes is indicated in parentheses. Asterisks indicate significant changes as determined by a two-tailed Mann-Whitney test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). (D) Superimposed pairwise correlation analyses of the mean ratio changes of all genes (gray), IL-1α-regulated genes (blue), and gene sets significantly up- or down-regulated by siRNA knockdown (red). Ratio values from RELA knockdown conditions were compared with the knockdown of a RELA interactor in each case. Genes that are jointly regulated by knockdown of RELA and one of its interactors correspond to the Venn diagrams of (B) and are marked in red. Coefficients of correlation (Pearson’s r), corresponding p values and coefficients of determination (r 2 ) rare indicated for all comparisons. The complete set of data is provided in Supplementary Table 3.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Knockdown, Transfection, Luciferase, Control, Incubation, Microarray, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to project the protein interactions of all target genes defined by knockdowns of p65 / RELA or its interactors in IL-1α-stimulated cells into combined functional networks. (B) Table summarizing the numbers of mapped IDs (= nodes) corresponding to the gene groups shown in , their protein interactions (= edges) and the protein interaction network enrichment p values as derived from STRING. (C) Cytoscape-derived PPI networks. Nodes are colored and arranged according to the deregulation of the corresponding genes by knockdown of p65 / RELA or its interactors. Edges visualize known protein interactions, including the small number of interactions reported for p65 / RELA, S100A8 / 9, and TFE3 / TFEB. No interactions were found for ZBTB5 and GLIS2.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Functional Assay, Derivative Assay, Knockdown

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to use p65 / RELA ChIPseq data for delineating chromatin recruitment of RELA together with its interactors on the basis of DNA motifs and three possible scenarios of interactions. (B) Windows of 1000 base pairs surrounding experimentally determined p65 / RELA ChIPseq peaks were searched for motifs of RELA and REL using matrices from the JASPAR data base. P values indicated significant enrichment compared to the whole genome. The Venn diagram shows the overlap and inserts show motif compositions. (C) Venn diagrams indicating the overlap of motifs found for RELA or the RELA interactors TFE3, TFEB or GLIS2 in chromosomal regions assigned to p65 / RELA ChIPseq peaks. P values indicated significant enrichment compared to the whole genome. Inserts show motif compositions. (D) All target genes that were significantly up- or downregulated under basal or IL-1α-stimulated conditions as shown in or were collected and were examined for their association with a p65 / RELA ChIPseq peak. The pie charts show the numbers of RELA, TFE3, TFEB and GLIS2 motifs detected in siRNA RELA target genes with an annotated p65 / RELA peak in their promoters or enhancers. (E) Overlap of all genes with a p65 / RELA peak in promoters or enhancers and at least one motif for the indicated transcription factors in IL_1a-stimulated conditions. (F) Genome browser view of the TNFAIP3 locus with p65 / RELA ChIPseq peaks, activated enhancers and promoters (H3K27ac), accessible chromatin (ATACseq) and mRNA production (RNAseq) before and after 1 h of IL-1α stimulation. Data sets were from GSE64224, GSE52470 and GSE134436 and are aligned to HG19 ( ; ). p65 / RELA binding regions of 1000 bp under p65 / RELA peaks and identified TF motifs are indicated by horizontal lines. (G) HeLa cells were left untreated or were starved for 24 h in HBSS. Half of the cells was treated with IL-1α (10 ng / ml) for 1 h before the end of the experiment. ChIP-qPCR was performed with the indicated antibodies or IgG controls and a primer pair covering the TNFAIP3 promoter region (marked with an arrow in ). Floating bar plots show percent input plus the mean of all values from three independent biological replicates performed with two technical replicates. The complete set of data is provided in Supplementary Table 4.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Binding Assay, ChIP-qPCR

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: Venn diagrams indicating the overlap of RELA motifs with motifs of ZBTB factors that were found by miniTurboID to interact with RELA, in chromosomal regions assigned to p65 / RELA ChIPseq peaks. P values indicated significant enrichment compared to the whole genome. Inserts show motif compositions.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques:

Journal: Nature Communications

Article Title: Dysregulated mast cell activation induced by diabetic milieu exacerbates the progression of diabetic peripheral neuropathy in mice

doi: 10.1038/s41467-025-59562-z

Figure Lengend Snippet: a , b TEM images of ER-mitochondria contacts in mast cells (yellow color depicted the mitochondria; red color depicted the ER) and related quantification. ER, endoplasmic reticulum; OMM, outer mitochondrial membrane. N = 6 per group. c , d In situ PLA monitoring of ER-mitochondria interactions as reflected by genetic modulation of IP3R1-VDAC1 complex and related quantification. The single antibody condition was used as the negative control (NC) of the PLA reaction. PLA, proximity ligation assay; IP3R1, inositol triphosphate receptor 1; VDAC1, voltage-dependent anion channel 1. N = 6 per group. e UMAP plots of DPN and TLA nerve tissues overlaid with GLUT1, GLUT2, GLUT3, and GLUT4 genes. GLUT, glucose transporter. f Double immunostaining of GLUT1 (red) and GLUT3 (green) in mast cells. g , h Relative mRNA expression of GLUT1 and GLUT3 in mast cells, revealed by qPCR. i Double immunostaining of p-ERK1/2 (green) and ERK1/2 (red) in mast cells. ERK, extracellular signal-regulated kinase. j Western blot (WB) assessment of the expressions of p-ERK1/2, t-ERK1/2, p-mTOR, and t-mTOR in mast cells. mTOR, mechanistic target of rapamycin. k , l Quantification of P-ERK/T-ERK and P-mTOR/T-mTOR ratio based on the WB results in Fig. . P-ERK refers to the phosphorylated ERK, T-ERK refers to the total ERK, P-mTOR refers to phosphorylated mTOR, and T-mTOR refers to the total mTOR. N = 6 per group. m – o WB assessment and related quantification of GLUT3 expression and ERK1/2 phosphorylation in mast cells. Mast cells were treated with either siRNA-GLUT3 or siRNA-negative control (NC). The protein expression levels of GLUT3 were normalized to GAPDH. N = 3 per group. p , q Relative expression level of CHOP and HSPA5 based on WB results in Fig. 7R. The protein expression levels were normalized to tubulin. CHOP, C/EBP homologous protein; HSPA5, heat-shock-protein family A member 5. N = 6 per group. r Immunoblots of the expression of CHOP and HSPA5 in mast cells. All data were expressed as mean ± SD. Statistical significance was determined using two-tailed unpaired Student’s t tests ( b , d , g , h , k , l , p , and q ) and one-way ANOVA followed by Dunnett’s multiple comparison tests ( n and o ).

Article Snippet: Then the PVDF membranes were incubated with primary antibodies against ERK1/2 (4695S, 1:1000, Cell Signaling Technology), p-ERK1/2 (9101S, 1:1000, Cell Signaling Technology), mTOR (2972S, 1:1000, Cell Signaling Technology), p-mTOR (2971S, 1:1000, Cell Signaling Technology), CHOP (2895S, 1:1000, Cell Signaling Technology), HSPA5 (3177S, 1:1000, Cell Signaling Technology), GAPDH (10494-1-AP, 1:10000, Proteintech) and Tubulin (14555-1-AP, 1:10000, Proteintech).

Techniques: Membrane, In Situ, Negative Control, Proximity Ligation Assay, Double Immunostaining, Expressing, Western Blot, Phospho-proteomics, Two Tailed Test, Comparison

Journal: Experimental Animals

Article Title: Abietic acid attenuates sepsis-induced lung injury by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway to inhibit M1 macrophage polarization

doi: 10.1538/expanim.22-0018

Figure Lengend Snippet: Abietic acid regulated macrophage gene expression and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. (A–E) RAW264.7 cells were pre-treated with different concentration of AA for 2 h, and then stimulated with 1 µ g/ml LPS for 24 h, levels of M1 macrophage markers ( CD16 (A) and iNOS (B)) and M2 macrophage markers ( CD206 (C) and Arg-1 (D)) were measured by qPCR, and levels of p-p65 and p65 protein expression by western blot (E). Data from three independent replicates for each test are plotted as (mean ± SD) as a column chart, and P values were calculated by analysis of variance. *** P <0.001 vs. Control group, # P <0.05, ## P <0.01 and ### P <0.001 vs. LPS group.

Article Snippet: The information of antibodies were showed as follows: anti-NF-kB p65 antibody (1:500, ab32536, Abcam, Cambridge, UK), anti-NF-kB p65 (phospho S536) antibody (1:500, ab76302, Abcam), Anti-beta actin antibody (1:2,000, ab8226, Abcam), Goat Anti-Rabbit IgG H&L (HRP) (1:3,000, ab6721, Abcam) and Goat Anti-Mouse IgG H&L (HRP) (1:3,000, ab6789, Abcam).

Techniques: Gene Expression, Concentration Assay, Expressing, Western Blot, Control

Journal: Experimental Animals

Article Title: Abietic acid attenuates sepsis-induced lung injury by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway to inhibit M1 macrophage polarization

doi: 10.1538/expanim.22-0018

Figure Lengend Snippet: Abietic acid blocked nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway in BALF of sepsis mice. 6 or 24 h after cecal ligation and puncture, mice were euthanized, BALF was collected, and levels of p-p65 and p65 protein were measured by western blot. Representative protein bands are shown on the left, and a statistical comparison of the gray values of the protein bands is shown on the right. P values were calculated by analysis of variance. *** P <0.001 vs. Sham group, and ### P <0.001 vs. CLP group.

Article Snippet: The information of antibodies were showed as follows: anti-NF-kB p65 antibody (1:500, ab32536, Abcam, Cambridge, UK), anti-NF-kB p65 (phospho S536) antibody (1:500, ab76302, Abcam), Anti-beta actin antibody (1:2,000, ab8226, Abcam), Goat Anti-Rabbit IgG H&L (HRP) (1:3,000, ab6721, Abcam) and Goat Anti-Mouse IgG H&L (HRP) (1:3,000, ab6789, Abcam).

Techniques: Ligation, Western Blot, Comparison

Journal: bioRxiv

Article Title: Hepatocyte FAM210A deficiency disrupts mitochondrial function and triggers juvenile steatosis with compensatory repair in adulthood

doi: 10.1101/2025.09.02.673111

Figure Lengend Snippet: a Representative TEM images of liver mitochondria in Ctrl and Fam210a HKO mice at 3 and 5 weeks old (Scale bar, 0.5 µm). b Relative mitochondrial DNA (mtDNA) copy number in the liver of Ctrl and Fam210a HKO mice at 3, and 5 weeks old (n = 4; mean ± s.e.m.; two-tailed, unpaired student’s t -test). c Western blot analysis of mitochondrial dynamics–related proteins in Ctrl and Fam210a HKO livers at 1, 3, and 5 weeks old. d-g Quantification of the ratio of L-OPA1/S-OPA1 ( d ), total OPA1 ( e ), DRP1 ( f ), and MFN2 ( g ) protein levels shown in ( c ) (n = 3; mean ± s.e.m.; two-tailed, unpaired student’s t -test). h Proximity ligation assay (PLA) between FAM210A-FLAG and OPA1, YME1L and OMA1 in Fam210a-flag overexpressed HepG2 cells (Scale bar, 5 µm). i Co-immunoprecipitation (Co-IP) of FAM210A-Myc and YME1L in Fam210a-myc overexpressed HepG2 cells (n = 3 independent experiments).

Article Snippet: Following antibodies were used: C18orf19 (FAM210A, Invitrogen, PA5-53146; 1:1000), β-Actin (Santa Cruz, sc-47778; 1:1000), OxPhos Rodent WB Antibody Cocktail (Invitrogen, 45-809-9; 1:2000), OPA1 (BD Biosciences, 612606; 1:1000), DRP1 (Proteintech, 12957-1-AP; 1:1000), Mitofusin 2 (MFN2, Cell Signaling, 9482; 1:1000), HSP90 (Proteintech, 13171-1-AP; 1:2000), YME1L (Proteintech, 11510-1-AP; 1:1000), FLAG (Proteintech, 20543-1-AP; 1:20000), HRP AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch, 115-035-003; 1:10000), HRP AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch, 111-035-003; 1:10000).

Techniques: Two Tailed Test, Western Blot, Proximity Ligation Assay, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Cell Death & Disease

Article Title: CEMIP, acting as a scaffold protein for bridging GRAF1 and MIB1, promotes colorectal cancer metastasis via activating CDC42/MAPK pathway

doi: 10.1038/s41419-023-05644-z

Figure Lengend Snippet: A , B The Progression Free survival (PFS, A ) and Overall Survival (OS, B ) of colon cancer patients were generated through TCGA (The Cancer Genome Atlas) database from http://www.sangerbox.com/ . C Transwell chamber migration and invasion of SW480 cells transfected with Flag-GRAF1(GRAF1-overexpression plasmid with Flag tag attached in N terminal of GRAF1) or Scramble plasmid (negative control). *** P < 0.001; **** P < 0.0001. Scale bar, 50 μm. D The interactions between endogenous CEMIP and endogenous GRAF1 were detected by Co-IP in HCT116 cells. E , F The interactions between exogenous CEMIP and exogenous GRAF1 were detected by Co-IP in HEK293T cells transfected with the indicated constructs. G The interaction between endogenous GRAF1 and CEMIP in HCT116 cells was further confirmed by the proximity ligation assays (PLA). Scale bar, 10 μm.

Article Snippet: Then, the primary antibodies, GRAF1 (Proteintech, #17747-1-AP, 1:50 dilution) and CEMIP (Santa Cruz, #sc-293483,1:25 dilution), GRAF1 (Proteintech, #17747-1-AP, 1:50 dilution) and MIB1 (Santa Cruz, #sc-393551,1:25 dilution), CEMIP (Proteintech, #21129-1-AP, 1:50 dilution) and MIB1 (Santa Cruz, #sc-393551, 1:25 dilution) or IgG (Rabbit, Cell Signaling Technology, #3900, 1:5000 dilution) and IgG (Mouse, Cell Signaling Technology, #5415, 1:5000 dilution) were incubated with the cells for 2 h at 37 °C.

Techniques: Generated, Migration, Transfection, Over Expression, Plasmid Preparation, FLAG-tag, Negative Control, Co-Immunoprecipitation Assay, Construct, Ligation

Journal: Cell Death & Disease

Article Title: CEMIP, acting as a scaffold protein for bridging GRAF1 and MIB1, promotes colorectal cancer metastasis via activating CDC42/MAPK pathway

doi: 10.1038/s41419-023-05644-z

Figure Lengend Snippet: A Western blotting analysis of HCT116 cells transfected with shCEMIP (CEMIP-downregulated plasmid containing shRNA of CEMIP) and western blotting analysis of SW480 cells transfected with CEMIP-myc (CEMIP-upregulated plasmid containing CEMIP cDNA with c-myc tag attached in C terminal of CEMIP). B Western blotting analysis of CEMIP and GRAF1 in HCT116 cells transfected with the indicated plasmids with or without MG132 (26 S proteasome inhibitor, 10 μM, 6 h). C Western blotting analysis of the ubiquitination level of GRAF1 derived from Co-IP in HCT116 cells transfected with the indicated constructs. D HCT116 cells transfected with the indicated plasmid were treated with CHX (cycloheximide, 20 μg/mL) for the indicated times, and the expressions of GRAF1 were analyzed by western blotting analysis, * P < 0.05; ** P < 0.01. E Representative immunohistochemical staining of CEMIP and GRAF1 in human primary colorectal carcinoma. Scale bar, 50 μm. Statistical analysis of immunohistochemical staining of two proteins. F Western blotting analysis of the ubiquitination level of GRAF1 and GRAF1 (∆SH3) derived from Co-IP in HCT116 cells transfected with the indicated constructs. G HCT116 cells transfected with the indicated plasmids were treated with CHX (20 μg/mL) for the indicated times, and the expressions of GRAF1 and GRAF1 (∆SH3) were analyzed by western blotting analysis. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Then, the primary antibodies, GRAF1 (Proteintech, #17747-1-AP, 1:50 dilution) and CEMIP (Santa Cruz, #sc-293483,1:25 dilution), GRAF1 (Proteintech, #17747-1-AP, 1:50 dilution) and MIB1 (Santa Cruz, #sc-393551,1:25 dilution), CEMIP (Proteintech, #21129-1-AP, 1:50 dilution) and MIB1 (Santa Cruz, #sc-393551, 1:25 dilution) or IgG (Rabbit, Cell Signaling Technology, #3900, 1:5000 dilution) and IgG (Mouse, Cell Signaling Technology, #5415, 1:5000 dilution) were incubated with the cells for 2 h at 37 °C.

Techniques: Western Blot, Transfection, Plasmid Preparation, shRNA, Ubiquitin Proteomics, Derivative Assay, Co-Immunoprecipitation Assay, Construct, Immunohistochemical staining, Staining

Journal: Cell Death & Disease

Article Title: CEMIP, acting as a scaffold protein for bridging GRAF1 and MIB1, promotes colorectal cancer metastasis via activating CDC42/MAPK pathway

doi: 10.1038/s41419-023-05644-z

Figure Lengend Snippet: A Potential E3 ubiquitin ligases of GRAF1 were predicted by the UbiBrowser database. B Western blotting analysis of the predicted ligases derived from Co-IP in HCT116 cells transfected with the indicated constructs. C The interaction between endogenous GRAF1 and endogenous MIB1 was detected by Co-IP in HCT116 cells. Rabbit IgG was used as a negative control. D The interaction between exogenous GRAF1 and exogenous MIB1 was detected by Co-IP in HEK293T cells transfected with the indicated constructs. E The putative binding domain between GRAF1 and MIB1 predicted by the database mentioned above from ( http://ubibrowser.bio-it.cn/ubibrowser/strict/index/edgeinfo/sub/Q9UNA1/e3/Q86YT6 ). F The interaction between exogenous GRAF1 (∆SH3) and endogenous MIB1 were detected by Co-IP in HCT116 cells. G The interaction between endogenous GRAF1 and MIB1 was further confirmed by the PLA assay in HCT116 cells. Scale bar, 10 μm. H The protein level of CEMIP and GRAF1 detected by western blotting transfected with MIB1 plasmid (MIB1-upregulated plasmid containing MIB1 cDNA) or si-MIB1 (small interfering RNA of MIB1) in HCT116 cells. I Western blotting analysis of the ubiquitination level of GRAF1 derived from Co-IP in HCT116 cells transfected with the indicated constructs.

Article Snippet: Then, the primary antibodies, GRAF1 (Proteintech, #17747-1-AP, 1:50 dilution) and CEMIP (Santa Cruz, #sc-293483,1:25 dilution), GRAF1 (Proteintech, #17747-1-AP, 1:50 dilution) and MIB1 (Santa Cruz, #sc-393551,1:25 dilution), CEMIP (Proteintech, #21129-1-AP, 1:50 dilution) and MIB1 (Santa Cruz, #sc-393551, 1:25 dilution) or IgG (Rabbit, Cell Signaling Technology, #3900, 1:5000 dilution) and IgG (Mouse, Cell Signaling Technology, #5415, 1:5000 dilution) were incubated with the cells for 2 h at 37 °C.

Techniques: Ubiquitin Proteomics, Western Blot, Derivative Assay, Co-Immunoprecipitation Assay, Transfection, Construct, Negative Control, Binding Assay, Plasmid Preparation, Small Interfering RNA

Journal: Cell Death & Disease

Article Title: CEMIP, acting as a scaffold protein for bridging GRAF1 and MIB1, promotes colorectal cancer metastasis via activating CDC42/MAPK pathway

doi: 10.1038/s41419-023-05644-z

Figure Lengend Snippet: A The interactions between endogenous CEMIP and endogenous MIB1 were detected by Co-IP in HCT116 cells. Rabbit IgG was used as a negative control. B The interaction between exogenous CEMIP and exogenous MIB1 was detected by Co-IP in HEK293T cells. C The interactions between exogenous CEMIP deletion mutants and exogenous MIB1 were detected by Co-IP in HEK293T cells transfected with the indicated constructs. D The interaction between endogenous CEMIP and MIB1 was further confirmed by the PLA assay in HCT116 cells. Scale bar, 10 μm. E Western blotting analysis of proteins derived from Co-IP in HCT116 (left) and SW480 cells (right) transfected with the indicated plasmids. F Western blotting analysis of the ubiquitination level of GRAF1 derived from Co-IP in HCT116 cells transfected with the indicated constructs.

Article Snippet: Then, the primary antibodies, GRAF1 (Proteintech, #17747-1-AP, 1:50 dilution) and CEMIP (Santa Cruz, #sc-293483,1:25 dilution), GRAF1 (Proteintech, #17747-1-AP, 1:50 dilution) and MIB1 (Santa Cruz, #sc-393551,1:25 dilution), CEMIP (Proteintech, #21129-1-AP, 1:50 dilution) and MIB1 (Santa Cruz, #sc-393551, 1:25 dilution) or IgG (Rabbit, Cell Signaling Technology, #3900, 1:5000 dilution) and IgG (Mouse, Cell Signaling Technology, #5415, 1:5000 dilution) were incubated with the cells for 2 h at 37 °C.

Techniques: Co-Immunoprecipitation Assay, Negative Control, Transfection, Construct, Western Blot, Derivative Assay, Ubiquitin Proteomics

Journal: Cell Death & Disease

Article Title: CEMIP, acting as a scaffold protein for bridging GRAF1 and MIB1, promotes colorectal cancer metastasis via activating CDC42/MAPK pathway

doi: 10.1038/s41419-023-05644-z

Figure Lengend Snippet: A Transwell chamber migration and invasion of SW480 cells transfected with the indicated constructs. ** P < 0.01; *** P < 0.001; **** P < 0.0001. B Representative images of livers of BALB/c nude mice fixed in 10% formaldehyde. Numbers of liver metastases were detected in each group (day 28, n = 5). Representative HE staining of liver tissues was shown. Scale bar, 1 mm. ** P < 0.01; *** P < 0.001. C Body weight curves of mice. * P < 0.05; ** P < 0.01; *** P < 0.001. D Survival curves of mice. * P < 0.05; ** P < 0.01. E Representative immunohistochemical staining for CEMIP, GRAF1 in primary colorectal cancer tissues of BALB/c nude mice mentioned above. The group of GRAF1 were not founded in metastatic foci. Scale bar, 50 μm. F Transwell chamber migration and invasion of SW480 cells transfected with the indicated constructs. *** P < 0.001; **** P < 0.0001.

Article Snippet: Then, the primary antibodies, GRAF1 (Proteintech, #17747-1-AP, 1:50 dilution) and CEMIP (Santa Cruz, #sc-293483,1:25 dilution), GRAF1 (Proteintech, #17747-1-AP, 1:50 dilution) and MIB1 (Santa Cruz, #sc-393551,1:25 dilution), CEMIP (Proteintech, #21129-1-AP, 1:50 dilution) and MIB1 (Santa Cruz, #sc-393551, 1:25 dilution) or IgG (Rabbit, Cell Signaling Technology, #3900, 1:5000 dilution) and IgG (Mouse, Cell Signaling Technology, #5415, 1:5000 dilution) were incubated with the cells for 2 h at 37 °C.

Techniques: Migration, Transfection, Construct, Staining, Immunohistochemical staining