Journal: Molecular & cellular proteomics : MCP

Article Title: Identification of novel proteins associated with both alpha-synuclein and DJ-1.

doi: 10.1074/mcp.M600182-MCP200

Figure Lengend Snippet: FIG. 4. Affinity purification and WB validation of mortalin, grp94, calnexin, clathrin, and nucleolin in -synuclein and DJ-1 protein complexes. MES cells were treated with 20 nM rotenone (R) or DMSO (D) for 3 days and then lysed in Nonidet P-40 lysis buffer, respectively. The total cell lysate was purified with -synuclein or DJ-1 affinity column. Afterward equal amounts of protein (20 g) from the total cell lysate (input) and elutes were analyzed by WB with antibodies against -synuclein, DJ-1, mortalin, grp94, calnexin, clathrin, or nucleolin, respectively.

Article Snippet: Antibodies used include: -synuclein for Western blot (WB) (BD Pharmingen), -synuclein for IP (Cell Signaling Technology, Beverly, MA), -synuclein for immunofluorescent staining (Chemicon, Temecula, CA), DJ-1 (Novus Biologicals, Littleton, CO), mortalin/grp75 (Stressgen, San Diego, CA), nucleolin (Novus Biologicals), calnexin (BD Pharmingen), grp94 (Stressgen), and clathrin (Chemicon).

Techniques: Affinity Purification, Biomarker Discovery, Lysis, Purification, Affinity Column

Journal: Molecular & cellular proteomics : MCP

Article Title: Identification of novel proteins associated with both alpha-synuclein and DJ-1.

doi: 10.1074/mcp.M600182-MCP200

Figure Lengend Snippet: FIG. 5. Co-localization of mortalin, nucleolin, calnexin, grp94, and clathrin with -synuclein and DJ-1 in MES cells. MES cells were fixed and triple stained with antibodies against -synuclein (Column A, red); DJ-1 (Column B, green); and mortalin, nucleolin, calnexin, grp94, or clathrin (Column C, blue) simultaneously. The images were visualized with a confocal microscope. Merged im- ages are shown in white when three antibodies are co-localized (Column D).

Article Snippet: Antibodies used include: -synuclein for Western blot (WB) (BD Pharmingen), -synuclein for IP (Cell Signaling Technology, Beverly, MA), -synuclein for immunofluorescent staining (Chemicon, Temecula, CA), DJ-1 (Novus Biologicals, Littleton, CO), mortalin/grp75 (Stressgen, San Diego, CA), nucleolin (Novus Biologicals), calnexin (BD Pharmingen), grp94 (Stressgen), and clathrin (Chemicon).

Techniques: Staining, Microscopy

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: eLife

Article Title: Long non-coding RNA Neat1 and paraspeckle components are translational regulators in hypoxia

doi: 10.7554/elife.69162

Figure Lengend Snippet: Figure 6. p54nrb-interacting proteins, nucleolin and RPS2, control the FGF1 IRES activity. (A–C) Quantification of RPS2 (A), hnRNPM (B) and nucleolin (C) RNA expression in HL-1 cells transfected with siRNAs against Rps2, hnRNPM or nucleolin mRNA, respectively. RNA expression was measured by RT- qPCR and normalized to control siRNA. One representative experiment is shown with n=3 biological replicates. Student two-tailed t-test was performed with n=3 or Mann-Whitney test with n=9; *p<0.05, **p<0.01, ***<0.001, ****p<0.0001. (D) Capillary Simple Western of nucleolin following nucleolin knock-down. The full raw unedited gel is provided in Figure 6—figure supplement 1A (Figure 6—figure supplement 1—source data 1). (E) FGF1 IRES activity with knock-down by siRNA interference of candidate ITAF nucleolin in HL-1 in normoxia or hypoxia 1% O2 was performed as in Figure 2. The IRES activity values have been normalized to the control siRNA. Histograms correspond to means ± standard deviation of the mean, with a non- parametric Mann-Whitney test *p<0.05, **p<0.01. The mean has been calculated with nine cell culture biological replicates, each of them being already the mean of three technical replicates (27 technical replicates in total but the M-W test was performed with n=9). Detailed values of biological replicates are presented in Supplementary file 6. (F) Capillary Simple Western of endogenous FGF1 following nucleolin knock-down. Histograms correspond to means ± standard deviation. The source data or capillary Simple Western are provided in Figure 1—figure supplement 1B (Figure 6—figure supplement 1—source data 1).

Article Snippet: Capillary Western Diluted protein lysate was mixed with fluorescent master mix and heated at 95 °C for 5 min. Three μL of protein mix (1 mg/mL maximal concentration) containing Protein Normalization Reagent, blocking reagent, wash buffer, target primary antibody (rabbit anti- eIF2α [Cell Signaling Technology 9721]) diluted 1:50, mouse anti- phospho- eIF2α [Cell Signaling Technology 2103] diluted 1:50, mouse antip21 antibody [Santacruz, sc- 6246] diluted 1:50, rabbit anti- P54nrb diluted 1:200 [Santacruz, sc- 67016], rabbit anti- PSPC1 diluted 1:100 [bethyl laboratory, A303- 205A], mouse anti- SFPQ diluted 1:100 [Abcam, Ab11825]; rabbit anti- FGF1 diluted 1:25 [Abcam Ab207321], rabbit anti- Nucleolin diluted 1:50 [Novus biological, NB600- 241], secondary- HRP (ready to use rabbit or mouse ‘detection module’, DM- 001 or αDM- 002), and chemiluminescent substrate were dispensed into designated wells in a manufacturer- provided microplate.

Techniques: Control, Activity Assay, RNA Expression, Transfection, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Simple Western, Knockdown, Standard Deviation, 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: 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

Journal: Experimental & Molecular Medicine

Article Title: (Non)canonical Wnt signaling, cytoarchitecture and stemness: new insights from primary nonmetastatic, primary metastatic, regional and distant metastatic models of adrenocortical carcinoma

doi: 10.1038/s12276-025-01507-z

Figure Lengend Snippet: a Cellular localization of vimentin in all three models. b – d The localization of E-cadherin and N-cadherin in TVBF-7 ( b ) NCI-H295R ( c ) and MUC-1 ( d ) spheroids. e – g The cellular localization of MMP9 ( e ) Nucleolin ( f ) and Lamin B1–β-catenin–DAPI ( g ) in tumor spheroids of all three models. Scale bar, 50 μm.

Article Snippet: Nucleolin , STJ140144, St John’s Laboratory (London, UK) , 1/200.

Techniques: