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Figure Lengend Snippet: UV-induced NONO degradation is mediated by RNF8. ( A ) RNF8 depletion stabilizes NONO after UV damage. HeLa cells were transfected with control siRNA or RNF8 siRNA, and treated with UV (30 J/m 2 ) for 6 h. Cell lysates were processed for immunoblotting with the indicated antibodies. ( B ) UV irradiation increases the polyubiquitination of NONO. HeLa cells were irradiated with UV (30 J/m 2 ) for the indicated time points and processed for the in vivo ubiquitination assay (left panel). Immunoprecipitation experiment revealed that NONO interacted with SFPQ under the immunoprecipitation buffer condition containing 0.5% NP-40 (right panel). ( C ) UV-irradiation specifically ubiquitinates NONO but not SFPQ or PSPC1. In vivo ubiqutination assays of NONO, SFPQ and PSPC1 were performed with HeLa cells at the indicated time points following UV (30 J/m 2 ) irradiation. ( D ) Knockdown of RNF8 impairs in vivo NONO ubiquitination after UV DNA damage. HeLa cells transfected with control siRNA or RNF8 siRNA were treated with UV (30 J/m 2 ) for 4 h and processed for the in vivo ubiquitination assay. The ubiquitin-conjugated endogenous NONO was subsequently detected by Western blotting analysis using anti-Ub antibody.

Article Snippet: The following antibodies were used for Western blotting: anti-RNF8 (B-2) (#sc-271462, 1:5000), anti-GFP (#sc-9996, 1:3000), anti-Ub (A-5) (#sc-166553, 1:1000), anti-CDC25A (F-6) (#sc-7389, 1:1000), anti-p53 (#sc-126, 1:1000) and anti-HSP90 (H-114) (#sc-7479, 1:2000) from Santa Cruz Biotechnology; anti-phospho H2AX S139 (#ab11174, 1:20000), anti-H2AX (#ab11175, 1:10 000) and anti-TOPBP1 (#ab2402, 1:5000) from Abcam; anti-phospho CHK1 S345 (#2348, 1:2500), anti-CHK1 (#sc-8408, 1:3000) and anti-MYC (#2276, 1:2000) from Cell Signaling Technology; anti-FLAG (#F3165, 1:10 000), anti-biotin (#B7653, 1:10 000) and anti-β-actin (#A2228, 1:10000) from Sigma; anti-NONO (#611279, 1:10000) from BD Transduction Laboratories; anti-T7 (#69522, 1:10000) from Novagen; anti-HA (#clone 12CA5, 1:1000) from Roche; anti-ATRIP (#A300-095A, 1:2000), anti-XPC (#A301-122A, 1:2000) and anti-SFPQ (#A301-320A, 1:2000) from Bethyl Laboratories, Inc.; anti-K48-specific ubiquitin (#clone Apu2.07, 1:1000) and anti-K63-specific ubiquitin (#clone Apu3.A8, 1:1000) from Genentech Inc.

Techniques: Transfection, Western Blot, Irradiation, In Vivo, Ubiquitin Assay, Immunoprecipitation

Journal: Nature Communications

Article Title: Spatiotemporal-resolved protein networks profiling with photoactivation dependent proximity labeling

doi: 10.1038/s41467-022-32689-z

Figure Lengend Snippet: a Schematic of miniSOG-mediated BRD4 proximity labeling with irradiation time: 2, 5, 10, and 20 min. b Overlapping of the identified proteins for different illumination time points. The enrichment of the identified proteins is statistically significant in HEK293T-miniSOG-BRD4 compared with wild-type HEK293T. c The ion intensity in label-free quantification for representative known BRD4 binding proteins across the indicated irradiation times. n = 3 biologically independent samples. Data are presented as mean values ± SD. d Gene ontology (GO) analysis of proteins identified in 2 min group. The top ten GO terms are listed. Bubbles are colored according to to GO term class and bubble size is proportional to the number of proteins found in each term. e String analysis of BRD4 interacting proteins. Yellow circles are direct binders and gray circles are in the first layer of indirect binders. The red lines indicate experimentally determined interactions and cyan lines indicate predicted interactions. f Representative BRD4 binding targets identified in LC-MS/MS were validated using Western blot. g Co-immunoprecipitation experiments verified the interaction between SFPQ and BRD4. These experiments were independently repeated at least twice with similar results. Source data are provided as a Source Data file.

Article Snippet: Primary antibody anti-SFPQ rabbit monoclonal antibody (CST, #71992) was used at 1:1000 dilution in 5% non-fat milk in TBST and incubated overnight at 4 °C. anti-rabbit IgG was used at 1:5000 and incubated for 1 h at room temperature.

Techniques: Labeling, Irradiation, Quantitative Proteomics, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Immunoprecipitation

Journal: Neoplasia (New York, N.Y.)

Article Title: The FUS-DDIT3 Interactome in Myxoid Liposarcoma 1

doi: 10.1016/j.neo.2019.05.004

Figure Lengend Snippet: Reciprocal co-immunoprecipitation and proximity ligation assays validate FUS-DDIT3’s association with NONO, PSPC1 and SFPQ. (A) Whole cell lysates from two myxoid liposarcoma cell lines, 402-91 and 1765-92, each harboring a different variant of the FUS-DDIT3 fusion oncoprotein, were used to test for reciprocal co-immunoprecipitation of the Drosophila behavior/human splicing proteins NONO, PSPC1, and SFPQ with the FUS-DDIT3 oncoprotein. For color blots, FUS was detected in the green channel and DDIT3 in the red channel, and the FUS-DDIT3 band is indicated by an overlap of FUS and DDIT3 signal to form a merged yellow-colored band. Larger images of the same blots with visible IgG bands from each IP antibody are in Figure S4 to visualize the amount of antibody used. (B) Proximity ligation assay, a technique that amplifies a red signal when two proteins colocalize within 40 nm of each other in situ , was performed in myxoid liposarcoma cell lines 402-91, 1765-92, DL-221, and negative control cell line HeLa. Red signals indicate proximity between proteins of interest. Nuclei were counter-stained with DAPI (blue). Scale bar = 5 μm.

Article Snippet: Antibodies used for immunoprecipitations were: normal mouse or rabbit IgG (Santa Cruz), DDIT3 (L63F7) (Cell Signaling Technology, #2895), NONO (Abcam, ab70335), PSPC1 (Abcam, ab104238), and SFPQ (Novus Biologicals, NB-100-61045).

Techniques: Immunoprecipitation, Ligation, Variant Assay, Proximity Ligation Assay, In Situ, Negative Control, Staining