Journal:

Article Title: Reduction of XNkx2-10 expression leads to anterior defects and malformation of the embryonic heart

doi: 10.1016/j.mod.2006.07.008

Figure Lengend Snippet: Analysis of XNkx2-10 expression. (A) A cartoon indicating the differences in XNkx2-3, XNkx2-5 and XNkx2-10 expression patterns at developmental stages 24, 28 and 32. Expression regions were estimated from cardiac XNkx2 in situ hybridization analysis (Cleaver et al., 1996; Newman and Krieg, 1998; Newman et al., 2000; Sparrow et al., 2000). The first column delineates XNkx2-10 expression. Strong XNkx2-10 expression is indicated with royal blue while weak XNkx2-10 expression is indicated with baby blue. The second column represents an overlay compilation of XNkx2-3, XNkx2-5 and XNkx2-10 expression regions. XNkx2-3 expression is indicated in yellow while XNkx2-5 expression is indicated in red. Regions where both XNkx2-3 and XNkx2-5 expression overlap is indicated with the color orange. (B) The RT-PCR products of XNkx2-10 and EF1α. expression from stage 10.5 to 12 (lane 1), stage 22 (lane 2), stage 32 (lane 3), stage 46 (lane 4) and adult heart (lane 5). Lane 6 is a negative control (no reverse transcriptase). PCR products were collected at PCR cycle #45. (C) The RT-PCR products of XNkx2-3, XNkx2-5, XNkx2-10 and EF1α. expression from different Xenopus stage 46 and adult tissues. Lane 1 is the RT-PCR product from RNA harvested from Xenopus stage 46 embryo eyes. Lane 2 is from stage 46 embryo hearts. Lane 3 is from stage 46 embryo tails. Lane 4 is from the adult eye. Lane 5 is from an adult heart. Lane 6 is from an adult liver. Lane 7 is from an adult gut. Lane 8 is from adult skin. Lane 9 is from skeletal muscle. Lane 10 is a negative control that does not contain reverse transcriptase. PCR products were collect at PCR cycle #45. (D) Western blots with protein harvested from adult heart (lane 1) and skeletal muscle (lane 2). Lanes 3 and 4 provide evidence that we have generated an antibody that recognizes XNkx2-10. Lane 3 is XNkx2-10 protein generated in vitro. Primary antibodies were against nucleolin (95 kDa) and XNkx2-10 (black arrow) (29.2 kDa). Lane 4 is S-35-labeled XNkx2-10 generated in vitro.

Article Snippet: One piece was probed with the nucleolin primary antibody (DSHB #b6-6E7) ( Dreyer et al., 1985 ) and the other piece probed with either the XNkx2-10 antibody or T7 tag antibody.

Techniques: Expressing, In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Negative Control, Reverse Transcription, Western Blot, Generated, In Vitro, Labeling

Journal:

Article Title: Reduction of XNkx2-10 expression leads to anterior defects and malformation of the embryonic heart

doi: 10.1016/j.mod.2006.07.008

Figure Lengend Snippet: Reduction of XNkx2-10 leads to abnormal development. (A–G) Pictures of stage 22 embryos. Scale bar is 3 mm. (A) are non-injected embryos. (B) Embryos injected with 2 ng of XNkx2-10 mRNA (overexpression). (C) Morpholino control injected embryos. (D) Morpholino injected embryos. (E) Deed oligonucleotide injected embryos. (F) Embryos injected with both morpholino and 2 ng of XNkx2-10 mRNA (morpholino rescue). (G) Embryos injected with both Deed oligonucleotide and 2 ng of XNkx2-10 mRNA (Deed rescue), (a1) Stage 42 non-injected embryo. Scale bar is 3 mm. (b1) Stage 42 overexpression embryo, (c1) Stage 42 morpholino control injected embryo, (d1) Morpholino injected embryo. It has an abnormal head shape and delayed eye development (white arrows). (d2) Morpholino injected embryo that is stunted, has an abnormal head shape and delayed eye development, (e1) Deed oligonucleotide injected embryo that is stunted, has an abnormal head shape and delayed eye development. (e2) Deed injected embryo that is stunted, has an abnormal head shape, delayed eye development and delayed gut development, (f1) Stage 42 morpholino rescue embryo, (g1) Stage 42 Deed rescue embryo. (H) Western blot demonstrating that injection of either the morpholino or Deed oligonucleotide leads to reduced XNkx2-10 protein levels in stage 22 embryos (black arrows) and a Western blot demonstrating the translation of rescue mRNA (T7) in stage 22 embryos. (I) Western blot demonstrating XNkx2-10 and nucleolin protein levels at stage 36. (J) Western blot demonstrating XNkx2-10 and nucleolin protein levels at stage 46. Nucleolin serves as a positive control. The expected size of XNkx2-10 is 29.2 kDa while the expected size of nucleolin is 95 kDa. Lane 1 is protein from non-injected embryos. Lane 2 is protein from morpholino control injected embryos. Lane 3 protein from morpholino injected embryos. Lane 4 contains protein from Deed oligonucleotide injected embryos. Lane 5 contains protein from Deed rescue embryos.

Article Snippet: One piece was probed with the nucleolin primary antibody (DSHB #b6-6E7) ( Dreyer et al., 1985 ) and the other piece probed with either the XNkx2-10 antibody or T7 tag antibody.

Techniques: Injection, Over Expression, Control, Western Blot, Positive Control

Journal: Molecular and Cellular Biology

Article Title: ARID1a-DNA Interactions Are Required for Promoter Occupancy by SWI/SNF

doi: 10.1128/MCB.01008-12

Figure Lengend Snippet: The ARID domain of ARID1a is required for BAF-A occupancy at THBS1. (A) Mouse/human VISTA alignment of the THBS1 promoter and 5′ transcribed region. Salmon-colored peaks denote evolutionarily conserved regions, whereas lavender peaks denote exons. The locations of ChIP amplicons within this interval are plotted. (B to H) Formaldehyde-cross-linked chromatin from wild-type and Arid1aV1068G/V1068G MEFs was immunoprecipitated with ARID1a, ARID1b, ARID2, BRG1, BRM, INI1/SNF5, or Pol II antibodies. DNA was amplified by quantitative PCR to determine if loss of ARID-DNA binding leads to changes in BAF-A occupancy across THBS1. An intergenic, nonconserved downstream region (located at approximately kb +20) and two unlinked promoter control elements (GAPDH and INS-1) were used as negative genomic controls. Data were plotted as the percentage of total input or chromatin bound. (I) Whole-embryo protein lysates from triplicate pooled samples were used to examine THBS1 protein (reduced, monomeric form) expression differences by Western blotting. An overexposed image of the Western blot was also included to further emphasize these expression differences. The constitutive nuclear matrix protein, nucleolin, was used as a loading control. (J) cDNA synthesized from RNA isolated from wild-type or Arid1aV1068G/V1068G MEFs was used in a quantitative PCR to examine THBS1 expression differences following transfection with mock (nontargeting control), BRG1, or BRM siRNA. (K) Normalized luciferase activity of the THBS1 −2.8 kb promoter-luciferase fragment cotransfected with 0.05 to 0.5 μg of wild-type or mutant HA-mARID1a expression plasmids into NIH 3T3 cells. Cells cotransfected with the empty luciferase vector (−luc) or THBS1 −2.8 kb promoter-luciferase fragment and with empty pcDNA expression plasmids served as negative and positive controls, respectively. (L) Normalized luciferase activity of THBS1 −2.8 kb and −0.48 kb promoter-luciferase reporter plasmids cotransfected with 0.25 μg of pcDNA only (−) or wild-type or mutant HA-mARID1a expression plasmids. Empty luciferase reporter plasmid was used as a negative control. (M) Summary model of ChIP and expression data. Error bars in panels B to H and in panel J represent the SEMs. Error bars in panels K and L represent the standard deviations. Significant differences were calculated using a two-tailed Student t test (*, P < 0.05).

Article Snippet: Western blotting was performed using standard procedures and the following antibodies: ARID1a (A301-040A or A301-041A; Bethyl Labs), ARID1b (sc-32762; Santa Cruz), ARID2 (A302-230A; Bethyl Labs), BRG1 (sc-17796; Santa Cruz), BRM (sc-6450; Santa Cruz), INI1/SNF5 (A301-087A; Bethyl Labs), BAF60a (611728; BD Biosciences), cullin-2 (ab1870; Abcam), THBS-1 (MS-421-P0; Thermo Scientific), hemagglutinin (HA) tag (A190-108A; Bethyl Labs), β-actin (ab8226; Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sigma), and nucleolin (A300-711A; Bethyl Labs).

Techniques: Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Binding Assay, Control, Expressing, Western Blot, Synthesized, Isolation, Transfection, Luciferase, Activity Assay, Mutagenesis, Plasmid Preparation, Negative Control, Two Tailed Test

Journal: Cell reports

Article Title: mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching

doi: 10.1016/j.celrep.2023.112868

Figure Lengend Snippet: (A) MEFs stably expressing HA-eIF3D proteins were treated with/without Torin1 (250 nM) for 24 h, and RNA immunoprecipitation and qRT-PCR were performed. Data are the mean ± SD of three technical replicates. Statistical significance (*p < 0.05) was assessed by t test. (B) eIF3D interacting RBPs identified by mass spectrometry. (C–E) MEFs stably expressing control, HA-hnRNPF (C), HA-hnRNPK (D), and HA-SSB (E) were treated with Torin1 for 24 h, and their interacting proteins were identified and analyzed. (F–K) Stably knocked down MEFs with indicated shRNAs were treated with/without Torin1 for 24 h, and immunoblot blot analysis was performed. (L) RBP knockdown MEFs expressing HA-eIF3D were treated with Torin1 for 24 h, and RNA immunoprecipitation and qRT-PCR were performed. Data are the mean ± SD of three technical replicates. Statistical significance (*p < 0.05) was assessed by t test. (M) Stably knocked-down MEFs with indicated shRNAs were treated with/without Torin1 for 24 h, and qRT-PCR was performed. Data are the mean ± SD of three technical replicates. Statistical significance (*p < 0.05) was assessed by t test. See also .

Article Snippet: Anti-Stat3, anti-hnRNPK, and anti-hnRNPF antibodies were obtained from Proteintech.

Techniques: Stable Transfection, Expressing, RNA Immunoprecipitation, Quantitative RT-PCR, Mass Spectrometry, Control, Western Blot, Knockdown

Journal: Cell reports

Article Title: mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching

doi: 10.1016/j.celrep.2023.112868

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Stat3, anti-hnRNPK, and anti-hnRNPF antibodies were obtained from Proteintech.

Techniques: Recombinant, Software, Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Single-cell communication networks. (A) Incoming communication patterns of target cells, showing pathways to which each cell type responds. (B) Outgoing communication patterns of secreting cells, illustrating the pathways through which cells send signals, MIF, MK and CXCL pathway exhibit high activity. (C) Network diagram showing the strength of intercellular communication, with connections between various cell types. (D) Scatter plot comparing outgoing and incoming communication strengths across cell populations, with bubble size indicating the number of interactions, malignant cells have higher strength of intercellular communication. (E) Chord diagram depicting communication via the MK pathway between different cell types. (F) Ligand-receptor interaction probabilities within the MK pathway between malignant and other cell types. Dot size represents significance (P-value), and color represents communication probability highlighting the MDK-NCL signaling pathway. (G) Violin plots of MK pathway gene expression levels across cell types, showing gene activity variations, MDK has advancer expression level in malignant cells.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Activity Assay, Gene Expression, Expressing

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Spatial transcriptomics and MDK-NCL signal communication. (A) Niche clustering in spatial transcriptomics samples, identifying distinct ecological zones. (B) Spatial expression of representative markers in key regions: MUC1 (tumor region), LYZ (immune region), COL14A1 (stromal region), and SFTPC (normal region). (C) Violin plots displaying the expression of MUC1, LYZ, COL14A1, and SFTPC across different niches. (D) MCPcounter analysis showing the infiltration of six cell types (e.g., endothelial cells, fibroblasts, immune lineages) across spatial regions. (E) Spatial niche classification, distinguishing tumor, immune-stromal, and normal regions. (F) MDK-NCL ligand-receptor interaction analysis, spatially mapping MDK ligands, NCL receptors, and their binding regions.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Expressing, Binding Assay

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Single-cell pseudotime analysis. (A) Pseudotime trajectory analysis showing the 6 differentiation states of cells. (B) Subtype classification of malignant cells along the pseudotime trajectory. (C) Pseudotime scores mapped along the differentiation trajectory. (D) UMAP plot visualizing pseudotime scores across individual cells. (E) Box plots comparing pseudotime scores across different malignant cell clusters, cluster 0, 1, and 5 had higher pseudotime scores. (F) UMAP plot of differentiation states, with colors representing distinct states. (G) Stacked bar plots showing the proportion of differentiation states within each malignant cell cluster, cluster 0, 1, and 5 have larger proportion of state 6. (H) Expression dynamics of MK pathway genes (e.g., MDK, NCL, ITG genes) along the pseudotime trajectory, highlighting gene expression changes during differentiation, MDK and NCL express more in the later time.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Expressing, Gene Expression

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Association of MDK-NCL with the immune microenvironment. (A) Boxplot shows the expression levels of MDK and NCL genes in tumor and control groups, it exhibit higher activity in tumor group. (B) MDK-NCL enrichment scores in tumor and control groups. (C) Relative mRNA expression levels of MDK and NCL in tumor and control groups from in-house data. (D) Relative protein expression levels of MDK and NCL in tumor and control groups from in-house data. (E) Comparison of MDK protein expression levels between tumor and control groups. (F) Comparison of NCL protein expression levels between tumor and control groups. (G) Correlation of MDK and NCL expression with ImmuneScore, StromalScore, ESTIMATEScore, and TumorPurity. (H) Scatter plots depicting the relationship between MDK and NCL expression and immune-related scores (ImmuneScore, StromalScore, ESTIMATEScore) as well as TumorPurity. (I) Comparison of immune cell infiltration scores across high and low MDK-NCL expression groups for 28 immune cell types. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Expressing, Control, Activity Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Integrative single-cell and spatial transcriptomics analysis reveals MDK-NCL pathway’s role in shaping the immunosuppressive environment of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1546382

Figure Lengend Snippet: Association of MDK-NCL with immunotherapy response. (A) Comparison of tumor mutation burden (TMB) between high and low MDK-NCL expression groups. (B) Comparison of microsatellite instability (MSI) between high and low MDK-NCL groups. (C) Comparison of dysfunction scores between high and low MDK-NCL groups. (D) Comparison of exclusion scores between high and low MDK-NCL groups. (E) Expression of immunogenic cell death (ICD)-related genes in high and low MDK-NCL groups. (F) Expression levels of CTLA4 and PD1 in high and low MDK-NCL groups. (G) Comparison of immune checkpoint gene expression between high and low MDK-NCL expression groups. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Primary antibodies included MDK (1:1000, BM4392, BOSTER, Wuhan, China), NCL (1:1000, A00228-1, BOSTER, Wuhan, China), and GAPDH (1:1000, Sigma, USA), which was used as an internal control.

Techniques: Comparison, Mutagenesis, Expressing, Gene Expression

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: