Journal: Haematologica

Article Title: Accumulation of hypoxia-inducible factor-1 alpha protein and its role in the differentiation of myeloid leukemic cells induced by all-trans retinoic acid.

doi: 10.3324/haematol.13096

Figure Lengend Snippet: Figure 5. Specific suppression of Runx1, C/EBPα and PU.1 signifi- cantly prevents HIF-1α-triggered differentiation as well as its cooperation with ATRA. U937Tclone cells were stably transfected with a negative control (NC) or shRNA against Runx1 (shR-A2), C/EBPα (shR-C2), or PU.1 (shR-P3). The transfected cells were then incu- bated in the presence or absence of tetracycline and/or 10-8M of ATRA for 24 hours before blotting proteins as indi- cated (A) and for 3 days to detect CD11b+% cells (B).

Article Snippet: Western blot Cell lysates were fractionated on a 10% sodium dodecyl sulfate–polyacrylamide gel and immunoblots were performed with primary antibodies against HIF1α and HIF-1β (BD Transduction, San José, CA, USA), C/EBPα, Runx1 and PU.1 (Santa Cruz, CA, USA).

Techniques: Stable Transfection, Transfection, Negative Control, shRNA

Journal: Research

Article Title: Na + /K + -ATPase Modulates Purinergic P2X3 Receptor Function to Drive Bone Cancer Pain

doi: 10.34133/research.0932

Figure Lengend Snippet: Schematic diagram illustrating the proposed mechanism in the TCI model: injury-induced activation of P2X3R channels leads to Ca 2+ influx, which down-regulates protein phosphatase 2A (PP2A) expression and diminishes membrane localization of NKAα1. The decreased membrane NKAα1 disrupts its interaction with P2X3R, further promoting Ca 2+ influx and additional suppression of PP2A. Concurrently, reduced PP2A expression activates the extracellular signal-regulated kinase 1/2 (ERK1/2)–Runt-related transcription factor 1 (Runx1) signaling pathway, upregulating P2X3R expression. Together, these changes enhance the excitability of neurons and neuronal injury, which in turn promotes CCL5 release and glial cell activation, ultimately inducing bone cancer pain. SDH, spinal dorsal horn.

Article Snippet: The membranes were first blocked at room temperature for 1 h in Tris-buffered saline with 0.1% Tween-20 containing 5% nonfat milk and then incubated overnight at 4 °C with the following primary antibodies: NKAα1 (1:1,000, Invitrogen, MA3-928), P2X3R (1:1,000, Abcam, ab300493), phosphorylated ERK1/2 (1:1,000, CST, #9102), phosphorylated Runx1 (1:200, Santa Cruz, sc-293146), PP2Ac (1:1,000, ABclonal, A6702), β-actin (1:5,000, Proteintech, 20536-1-AP), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000, New Cell & Molecular Biotech, AB2100).

Techniques: Activation Assay, Expressing, Membrane

Journal: Journal of proteome research

Article Title: Analysis of oxygen/glucose-deprivation-induced changes in SUMO3 conjugation using SILAC-based quantitative proteomics

doi: 10.1021/pr200834f

Figure Lengend Snippet: Mass spectrometry-based quantification of SUMO conjugation via SILAC approach. . (A) Representative SILAC peptide pairs for an upregulated protein (RUNX1) and a downregulated protein (TIF1β). Peptide identifications are included in the figure, and associated m/z and intensity information in Table S1. (B) Non-normalized ratio plot including all quantified peptides in the experiment, with 2 peptides from RUNX1 and 31 peptides from TIF1β in black and peptides from the other 186 proteins in grey.

Article Snippet: Antibodies used in this study include anti-SUMO2/3 polyclonal antibody (Covance), anti-HA polyclonal antibody (Cell Signaling), anti-ubiquitin monoclonal antibody (Cell Signaling), anti-TIF1β polyclonal antibody (Cell Signaling), and anti-RUNX1 polyclonal antibody (Novus Biologicals).

Techniques: Mass Spectrometry, Conjugation Assay

Journal: Journal of proteome research

Article Title: Analysis of oxygen/glucose-deprivation-induced changes in SUMO3 conjugation using SILAC-based quantitative proteomics

doi: 10.1021/pr200834f

Figure Lengend Snippet: Verification of proteomic analysis. Proteomic analysis data were verified for RUNX1 (A) and TIF1β (B). HA-tagged SUMO3 conjugated proteins of extracts from control and OGD cultures were immunoprecipitated using anti-HA agarose and eluted with 100 μg/ml HA peptide. Eluates were left intact or incubated with SUMO protease to release SUMO from target proteins, loaded onto SDS-PAGE gels and immunoblotted using antibodies against RUNX1 (A) and TIF1β (B). WB, Western blotting; IP, immunoprecipitation.

Article Snippet: Antibodies used in this study include anti-SUMO2/3 polyclonal antibody (Covance), anti-HA polyclonal antibody (Cell Signaling), anti-ubiquitin monoclonal antibody (Cell Signaling), anti-TIF1β polyclonal antibody (Cell Signaling), and anti-RUNX1 polyclonal antibody (Novus Biologicals).

Techniques: Immunoprecipitation, Incubation, SDS Page, Western Blot