immunoprecipitation reactions  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: INCENP and CDCA8 predict neoadjuvant chemotherapy response and outcomes in esophageal squamous cell carcinoma

doi: 10.1038/s41467-026-68371-x

Figure Lengend Snippet: a , b Representative images and analysis of INCENP and CDCA8 protein expression levels after YTHDF3 knockdown in PDX-derived organoids (PDXO) by immunofluorescence. Scale bar = 10 μm. Mean protein fluorescence intensity was determined using ImageJ software. c , d Gene plots illustrating YTHDF3 binding to INCENP and CDCA8 mRNAs, as measured by RIP-seq. The normalized read distribution: input (yellow) and YTHDF3 (purple) along the mRNAs. e RNA immunoprecipitation-qPCR (RIP-qPCR) highlighting the indicated mRNA transcripts bound by YTHDF3 in ESCC cells. f , g Representative immunofluorescence staining of newly synthesized proteins and statistical analysis in KYSE30 and KYSE150 cells after YTHDF3 overexpression. Relative fluorescence intensity was determined using ImageJ software. Puromycin staining (green) indicates newly synthesized proteins; nuclei are stained with DAPI (blue). Scale bar = 50 μm. h , i Mock or YTHDF3 knockdown KYSE30 and KYSE150 cells were transfected with pmirGLO-INCENP or pmirGLO-CDCA8 reporters for 48 h. Translation efficiency of INCENP or CDCA8 is defined as the quotient of reporter protein production (F-luc/R-luc). j , k Co-immunoprecipitation of endogenous YTHDF3 and eIF3A in ESCC cells. Representative immunoblots shown in figures were repeated three times independently with similar results. l , m In situ detection and quantification of YTHDF3–eIF3A interactions in the indicated ESCC cell lines were measured using PLA assays, and puncta per cell were determined using ImageJ software. Scale bar = 10 μm, n = 5 biological replicates. n RIP-qPCR illustrating the association of eIF3A with the indicated transcripts in YTHDF3 knockdown KYSE30 cells. o RIP-qPCR illustrating the association of eIF3A with the indicated transcripts in Ebselen-treated KYSE30 cells. RIP-qPCR illustrating the association of YTHDF3 ( p ) and eIF3A ( q ) with the indicated transcripts in METTL3 knockdown KYSE30 cells. Data were analyzed by two-tailed unpaired t-test ( b , e , g , h , i , m ) or one-way ANOVA ( n , o , p , q ). n = 3 biological replicates ( b , e , g , h , i , n , o , p , q ). Data were presented as means ± SD. Source data are provided as a file.

Article Snippet: Ten percent (10%) of the lysate was stored as “input”, 80% was used in immunoprecipitation reactions with anti-YTHDF3 antibody (Abcam, ab220161), and the remaining 10% was incubated with rabbit IgG (Cell Signaling Technology) as a negative control and named “IgG”, respectively.

Techniques: Expressing, Knockdown, Derivative Assay, Immunofluorescence, Fluorescence, Software, Binding Assay, RNA Immunoprecipitation, Staining, Synthesized, Over Expression, Transfection, Immunoprecipitation, Western Blot, In Situ, Two Tailed Test

Journal: Cellular & Molecular Biology Letters

Article Title: CircNF1 modulates the progression and immune evasion of esophageal squamous cell carcinoma through dual regulation of PD-L1

doi: 10.1186/s11658-025-00712-y

Figure Lengend Snippet: CircNF1 blocks USP7 binding to ANXA1, leading to increased binding between USP7 and PD-L1. A Co-IP assays were performed using antibodies against USP7 and ANXA1 in KYSE150 cells. B Co-IP analysis of the interaction between USP7 and ANXA1 in circNF1-overexpressed KYSE150 cells and control cells. C Detection of ANXA1 polyubiquitination level in KYSE150 (left panel) and TE1 (right panel) cells transfected with si-USP7 or si-circNF1 using immunoprecipitation. D Detection of PD-L1 polyubiquitination level in KYSE150 (left panel) and TE1 (right panel) cells transfected with si-USP7 or overexpressed circNF1 using immunoprecipitation. E , F Co-IP analysis of the interaction between USP7 and PD-L1 in control and ANXA1-deficient KYSE150 (left panel) and TE1 (right panel) cells

Article Snippet: Each immunoprecipitation reaction was pulled down by magnetic protein A/G beads (MedChemExpress, Princeton, NJ, USA).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Control, Transfection, Immunoprecipitation

Journal: Cellular & Molecular Biology Letters

Article Title: CircNF1 modulates the progression and immune evasion of esophageal squamous cell carcinoma through dual regulation of PD-L1

doi: 10.1186/s11658-025-00712-y

Figure Lengend Snippet: Spatially competitive binding of ANXA1 by circNF1 decreases polyubiquitination modification of PD-L1. A Schematic representation of ANXA1 protein structure and its truncation constructs. B RIP assays validating the ectopic expression of Myc-tagged ANXA1 (full-length or short truncations) binding to circNF1 were presented using agarose gel electrophoresis (upper panel) or qRT-PCR (bottom panel) after immunoprecipitation in KYSE150 cells. C Co-IP revealing that the third repeat domain at the C-terminus of ANXA1 protein interacted with USP7 in KYSE150 cells. D Schematic representation of USP7 protein structure and its truncation constructs. E , F Co-IP showing the detailed region of USP7 bound to ANXA1 ( E ) or PD-L1 ( F ) in KYSE150 cells. G Co-IP detecting the antagonism of full-length or TRAF domain-deficient USP7 on upregulated endogenous PD-L1 ubiquitination caused by knocking down circNF1 in KYSE150 (left panel) and TE1 (right panel) cells. H Co-IP detecting the antagonism of full-length or TRAF domain-deficient USP7 on upregulated endogenous PD-L1 ubiquitination caused by ectopic expression of ANXA1 in KYSE150 (left panel) and TE1 (right panel) cells

Article Snippet: Each immunoprecipitation reaction was pulled down by magnetic protein A/G beads (MedChemExpress, Princeton, NJ, USA).

Techniques: Binding Assay, Modification, Construct, Expressing, Agarose Gel Electrophoresis, Quantitative RT-PCR, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Advanced Science

Article Title: Nuclear Condensates of WW Domain‐Containing Adaptor With Coiled‐Coil Regulate Mitophagy via Alternative Splicing

doi: 10.1002/advs.202406759

Figure Lengend Snippet: Mass spectrometry identified RBM12 as an interacting protein of eGFP‐WAC. a) A schematic diagram illustrating the immunoprecipitation–mass spectrometry procedure used in this study is shown. b) Heatmap of identified eGFP‐WAC‐interacting proteins. c) Mass spectrometry analysis of eGFP‐WAC immunoprecipitates. Selected peptide hits and unused scores are shown. A higher unused score means more credibility. d) Immunofluorescence assays were conducted to evaluate the colocalization of eGFP tag or eGFP‐tagged WAC and endogenous WAC with endogenous RBM12. The Pearson correlation coefficient was used to assess the overlap between each fluorescence signal in the nucleus region. Green boxes contain the 25th to 75th percentiles of the dataset. Black whiskers mark the 10th and 90th percentiles. Outliers are marked with black dots. Scale bar = 2 µm.

Article Snippet: The 10% lysate sample was stored and named “Input”, 80% was used in immunoprecipitation reactions with an anti‐RBM12 antibody (sc‐514259; Santa Cruz Biotechnology) and named “IP”, and 10% was incubated with normal mouse IgG (12‐371, Sigma) as a negative control and named “IgG”.

Techniques: Mass Spectrometry, Immunoprecipitation, Immunofluorescence, Fluorescence

Journal: Advanced Science

Article Title: Nuclear Condensates of WW Domain‐Containing Adaptor With Coiled‐Coil Regulate Mitophagy via Alternative Splicing

doi: 10.1002/advs.202406759

Figure Lengend Snippet: WAC interacts with the client protein RBM12 to cause co‐phase separation. a) Immunofluorescence images of HeLa cells transfected with 4 truncated versions of WAC with eGFP and endogenous RBM12. Nuclei were stained with 4’,6‐diamidino‐2‐phenylindole (DAPI). Scale bar = 2 µm. b) Diagram of 3 truncated domain maps of human RBM12 mutants. Human RBM12 contains 3 RNA recognition motifs (RRMs) and 1 proline‐rich linker region. c) Coexpression of 3 truncated versions of RBM12 with mCherry and eGFP‐WAC. Nuclei were stained with DAPI. Scale bar = 10 µm. d) In vitro condensate formation of purified full‐length or 4 truncated versions of eGFP–WAC (10 µM) mixed with 10 µ m WT mCherry–RBM12. Scale bar = 5 µm. e) The quantification of the droplets is shown in Figure . Droplets in each group were quantified (n = 5, two‐way ANOVA). f) The ability of HA‐tagged wild‐type WAC or WACΔCC to interact with Flag–RBM12 was examined using a coimmunoprecipitation assay with anti‐Flag antibodies via immunoprecipitation (IP), followed by western blotting with anti‐HA antibodies (top). The abundance of these proteins in the cell lysates was examined using western blotting (bottom). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. The results are from more than three independent experiments. Values are mean ± SD.

Article Snippet: The 10% lysate sample was stored and named “Input”, 80% was used in immunoprecipitation reactions with an anti‐RBM12 antibody (sc‐514259; Santa Cruz Biotechnology) and named “IP”, and 10% was incubated with normal mouse IgG (12‐371, Sigma) as a negative control and named “IgG”.

Techniques: Immunofluorescence, Transfection, Staining, In Vitro, Purification, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot