Journal: bioRxiv

Article Title: Different localization of fluorescently labeled N- and C-termini of nucleolin variants in human glioblastoma cell culture

doi: 10.1101/596916

Figure Lengend Snippet: 2D Western blot hybridization of the Sus\fP2 (A,B) and MSC (C,D) cell culture lysate. A – antibodies against the N-terminus of nucleolin (#N2662, Sigma, USA). B – antibodies against the C- terminus of nucleolin (#A300-711A, Bethyl, USA). The presence of multiple protein products recognized by antibodies against the N-terminus but not antibodies against the C-terminus is noticeable.

Article Snippet: Primary antibodies were used: anti-GFP in a titer of 1:8000 (AB011, Evrogen, Russia), anti-NCL in a titer of 1:7000 (#a300-711A, Bethyl, USA), anti-FLAG M2 in a titer of 1:8000 (#F3165-.2MG, Sigma, USA).

Techniques: Western Blot, Hybridization, Cell Culture

Journal: Scientific Reports

Article Title: Novel roles for LIX1L in promoting cancer cell proliferation through ROS1-mediated LIX1L phosphorylation

doi: 10.1038/srep13474

Figure Lengend Snippet: ( A ) Phosphorylated LIX1L was immunoprecipitated from the cytosolic and nuclear fractions of HEK-293FLG and HEK-293FLG-LIX1L cells using a FLAG antibody. Immunoprecipitates were analyzed through western blot analysis with a LIX1L antibody and phosphorylated serine-, threonine- and tyrosine-specific antibodies (upper panels). In the cytosolic and nuclear fractions of the HEK-293FLG and HEK-293FLG-LIX1L cells treated with 25 μM PY95 as a negative control or PY136, immunoprecipitates obtained using the FLAG antibody were analyzed through a western blot analysis with the LIX1L antibody and phosphorylated tyrosine-specific antibodies (bottom panels). Representative blots from HEK-293FLG and HEK-293FLG-LIX1L cell lines are shown. ( B ) The cell counts of HEK-293FLG and HEK-293FLG-LIX1L cells after treatment with PY136 (left panel). The HEK-293FLG and HEK-293FLG-LIX1L cells were cultured in semisolid methylcellulose media. The HEK-293FLG-LIX1L cells were left untreated or were treated with PY136 (25 μM). After 14 days in culture, colony formation was analyzed, and the cells were viewed using phase-contrast microscopy. The colonies formed from each cell type (3 × 10 2 to 5 × 10 2 cells/plate) were counted following plating onto semisolid methylcellulose media (right upper panel). Original magnification 4x (right bottom panels). These data are shown as the mean ± SD for independent experiments. **p < 0.05, *p < 0.01 . ( C ) The results of the immunoblot analysis of the cytosolic fraction treated with or without RNase in HEK-293FLG-LIX1L cells. The black arrow indicates the FLAG-LIX1L fusion protein. The red arrows indicate the detected proteins associated with the LIX1L-RNA complex. ( D ) Western blot analysis revealed that LIX1L interacted with the RIOK1, nucleolin and PABPC4 proteins in the cytoplasm of HEK-293 cells. In ( A ) and ( D ), the cropped blots were run under the same experimental condition.

Article Snippet: A Rabbit anti-LIX1L polyclonal antibody (Abnova, Taipei, Taiwan), mouse anti-FLAG monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phosphothreonine polyclonal antibody (Abcam, Cambridge, MA, USA), rabbit anti-phosphoserine polyclonal antibody (Abcam), rabbit anti-phosphotyrosine polyclonal antibody (Abcam), rabbit anti-RIOK1 polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-nucleolin polyclonal antibody (Origene, Rockville, MD, USA), rabbit anti-PABPC4 polyclonal antibody (Abnova), rabbit anti-Cofilin polyclonal antibody (Cell Signaling Technology (CST), Beverly, MA, USA), rabbit anti-phospho-Cofilin (Ser 3) polyclonal antibody (CST), rabbit anti-α Tubulin polyclonal antibody (Abcam), rabbit anti-Lamin A + C monoclonal antibody (Abcam), rabbit anti-ROS1 monoclonal antibody (CST) and anti-Actin antibody (Abcam) were used in the present study.

Techniques: Immunoprecipitation, Western Blot, Negative Control, Cell Culture, Microscopy

Journal: Scientific Reports

Article Title: Novel roles for LIX1L in promoting cancer cell proliferation through ROS1-mediated LIX1L phosphorylation

doi: 10.1038/srep13474

Figure Lengend Snippet: The identification of LIX1L-associated proteins by MALDI-TOF/TOF mass spectrometry.

Article Snippet: A Rabbit anti-LIX1L polyclonal antibody (Abnova, Taipei, Taiwan), mouse anti-FLAG monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phosphothreonine polyclonal antibody (Abcam, Cambridge, MA, USA), rabbit anti-phosphoserine polyclonal antibody (Abcam), rabbit anti-phosphotyrosine polyclonal antibody (Abcam), rabbit anti-RIOK1 polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-nucleolin polyclonal antibody (Origene, Rockville, MD, USA), rabbit anti-PABPC4 polyclonal antibody (Abnova), rabbit anti-Cofilin polyclonal antibody (Cell Signaling Technology (CST), Beverly, MA, USA), rabbit anti-phospho-Cofilin (Ser 3) polyclonal antibody (CST), rabbit anti-α Tubulin polyclonal antibody (Abcam), rabbit anti-Lamin A + C monoclonal antibody (Abcam), rabbit anti-ROS1 monoclonal antibody (CST) and anti-Actin antibody (Abcam) were used in the present study.

Techniques: Virus

Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells.

doi: 10.1016/j.bbagrm.2017.01.006

Figure Lengend Snippet: Fig. 1. Cytoplasmic form of nucleolin. (A) The N-terminal, middle domain, and C-terminal immunogens used to raise nucleolin antibodies. (B) Nucleolin immunoblot of cytoplasmic and nuclear fractions by antibodies raised against the N-terminal, middle domain, and C-terminal immunogens. All antibodies detect a single band with a MW ≅110 kDa in SDS-PAGE. (C) Actin, HDAC1, and Pan3 immunoblots show purity of cytoplasmic and nuclear lysates of Hey ovarian cancer cells. (D) Nucleolin immunoblot of cytoplasmic and nuclear fractions detects a single band with a pI ≅4.6 in IEF. (E) Subcellular localization of nucleolin by indirect immunofluorescence microscopy in MCF10A non-cancerous epithelial breast cells. The N-terminal raised antibody does not detect nucleolin in either the cytoplasm or nucleus. The middle domain raised antibody detects nucleolin both in the cytoplasm and nucleus. The C-terminal raised antibody detects nucleolin in the nucleus only. (F) Subcellular localization of nucleolin in MDA-MB-231 breast cancer cells. The N-terminal raised antibody detects nucleolin in the cytoplasm only. The middle domain raised antibody detects nucleolin both in the cytoplasm and nucleus. The C-terminal raised antibody detects nucleolin only in the nucleus, primarily in the nucleolus. Nucleolin shRNA decreased nucleolin signal in the cytoplasm detected by the N-terminal raised antibody. (G) The cytoplasmic form of nucleolin in MDA-MB-231 cells detected by the N-terminal and middle domain raised antibodies is co-localized. The bar is 10 μm.

Article Snippet: MycDDK-tagged nucleolin (Origene) was purchased.

Techniques: Western Blot, SDS Page, Microscopy, shRNA

Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells.

doi: 10.1016/j.bbagrm.2017.01.006

Figure Lengend Snippet: Fig. 3. Nucleolin interacts with the hl structure in the 5′UTR as well as G-quadruplex in the 3′UTR of CSF-1 mRNA. (A) RNA electrophoretic mobility shift assay shows an association of nucleolin (tagged with myc-DDK) with the hl structure from the 5′UTR as well as with the G-quadruplex from the 3′UTR of CSF-1 mRNA. G-quadruplex is used as a nucleolin binding control [17]. Nucleolin does not associate with either the linear 5′hl sequence or 5′UTR without hl sequence. (B) Luciferase protein level is increased (4– 5-fold) in wild type construct (Wt = Wt) in response to the nucleolin overexpression in SKOV3 cells. In contrast, luciferase protein level is not increased in the hl deletion construct {D(21) = Wt} in response to the nucleolin overexpression. (C) Luciferase mRNA stability is decreased in D(21) = Wt (red solid line) compared to Wt = Wt (black solid line) in SKOV3 cells. However, there was no significant effect of nucleolin overexpression on luciferase mRNA stability (broken lines). The cDNA was synthesized by random primer (pdN6). This suggests an effect of nucleolin on luciferase mRNA translation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: MycDDK-tagged nucleolin (Origene) was purchased.

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Control, Sequencing, Luciferase, Construct, Over Expression, Synthesized

Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells.

doi: 10.1016/j.bbagrm.2017.01.006

Figure Lengend Snippet: Fig. 4. Nucleolin forms mRNP complex with eIF4G and CSF-1 mRNA in Hey ovarian cancer cells. (A) N-terminal raised antibody (N-NCL) immunoprecipitates nucleolin only in RNases- treated sample. In the same sample, nucleolin co-immunoprecipitates with eIF4G. (B) Middle domain raised antibody (M-NCL) immunoprecipitates nucleolin in all treatments. However, nucleolin did not co-immunoprecipitate with eIF4G in the same samples. (C) C-terminal raised antibody (C-NCL) immunoprecipitates nucleolin. However, nucleolin did not co-immunoprecipitate with eIF4G in the same samples. (D) eIF4G antibody immunoprecipitates eIF4G in all treatments. However, eIF4G co-immunoprecipitates with nucleolin only in RNases-treated sample. (E) RT-qPCR measurements of CSF-1 mRNA in nucleolin or eIF4G immunoprecipitates of Hey cell lysates show direct interaction between either nucleolin or eIF4G and CSF-1 mRNA. After IP of RNA-protein complexes from Hey cell lysates, RNA was isolated and used in RT reactions and amplified by real time PCR. The graph shows relative mRNA levels in either nucleolin or eIF4G IP compared with control IgG IP conditions. The mean ± SD of CSF-1 mRNA normalized for GAPDH mRNA is depicted (n = 3). GAPDH mRNA was set to equal 1.

Article Snippet: MycDDK-tagged nucleolin (Origene) was purchased.

Techniques: Quantitative RT-PCR, Isolation, Real-time Polymerase Chain Reaction, Control

Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells.

doi: 10.1016/j.bbagrm.2017.01.006

Figure Lengend Snippet: Fig. 5. Cytoplasmic nucleolin co-localizes with the eIF4G in MDA-MB-231 breast cancer cells. The cytoplasmic nucleolin (green) detected by the N-terminal raised antibody co-localized with eIF4G (red). The bar is 10 μm.

Article Snippet: MycDDK-tagged nucleolin (Origene) was purchased.

Techniques:

Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: Expression of the cytoplasmic nucleolin for post-transcriptional regulation of macrophage colony-stimulating factor mRNA in ovarian and breast cancer cells.

doi: 10.1016/j.bbagrm.2017.01.006

Figure Lengend Snippet: Fig. 7. Formation of a nucleolin-mediated mRNP in translational regulation. Binding of nucleolin to the hl structure in the 5′UTR attracts eIF4F complex formation and enhances translation. Binding of nucleolin to the 3′UTR facilitates miRISC formation resulting in miRNA-directed deadenylation, but blocks further mRNA decay.

Article Snippet: MycDDK-tagged nucleolin (Origene) was purchased.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Dependency Map correlation analysis reveals WDR89 as a genome maintenance factor

doi: 10.64898/2026.01.28.698016

Figure Lengend Snippet: WDR89 is a nucleolar protein that responds to double-strand DNA break-inducing stresses. ATM-dependent nucleolar accumulation of WDR89 upon genotoxic stress. (A) WDR89 localizes predominantly in the nucleus and overlaps largely with nucleolin. (B) Quantification of WDR89 nuclear staining intensity following ionizing radiation. (C) Quantification of WDR89 nuclear staining intensity following genotoxic chemical treatments. (D) Confirmation of DNA damage by γH2AX foci staining of the conditions in C. (E) Effect of ATM inhibitor treatment on WDR89 nuclear intensity changes after IR. (F) Western blot of chromatin fractionation for WDR89 and the indicated markers. For panels in B, C, D and E, ****p<0.0001, by ANOVA with Tukey’s post-hoc test or by T-test where appropriate.

Article Snippet: Immunofluorescence staining was performed using anti-γ-H2AX antibodies (Abcam, ab81299, Santa Cruz, sc-517348), an anti-Nucleolin antibody (Novus, NBP2-44610), anti WDR89 antibody (Bethyl, A301-872A) or p53 antibody (Cell Signaling, #9282) at 1:250 dilution and Alexafluor 568 goat anti-rabbit secondary and Alexafluor 488 goat anti-mouse antibodies (Invitrogen) at 1:400 dilution.

Techniques: Staining, Western Blot, Fractionation