Journal: Nature Communications

Article Title: GPR4 promotes immune exclusion in colon cancer through LOXL2-mediated extracellular matrix remodeling

doi: 10.1038/s41467-025-67967-z

Figure Lengend Snippet: A GPR4 regulates JAK2 and STAT3 phosphorylation via western blotting. n = 3. Scale bar: 100 µm. B Representative IF images of STAT3-Y705 phosphorylation regulated by GPR4. C Quantitative analysis of STAT3-Y705 phosphorylation in GPR4 - OE SW480 cell line and ( D ) GPR4 - KD HCT116 cell lline. Data are presented as mean ± SD. Two-tailed t test, n = 3 biologically independent samples. E Static treatment reduces both pSTAT3-Y705 and the expression of LOXL2, COL1A1, and TGF-β in GPR4 - OE SW480 cells. F RT-PCR analysis for LOXL2 in SW480 cell lines after receiving Stattic treatment. Two-tailed t test, n = 3 biologically independent samples. G RT-PCR analysis for TGF-β in SW480 cell lines after receiving Stattic treatment. Data are presented as mean ± SD.Two-tailed t test, n = 3 biologically independent samples. H STAT3 transcription factor binding site. I In SW480 cells, STAT3 binds to the LOXL2 promoter region confirmed via ChIP assays. J STAT3 binds to the TGF-β promoter region confirmed via ChIP assays. K RT-PCR assesses the transcriptional activity of LOXL2 promoter regions and TGF-β promoter region L . Data are presented as mean ± SD. Two-tailed t test, n = 3 biologically independent samples. ns > 0.05.

Article Snippet: Subsequently, Transcription Factor Staining Kit (Elabscience, E-CK-A108) was applied for cells fixation and Permeabilization as recommended method.

Techniques: Phospho-proteomics, Western Blot, Two Tailed Test, Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: RREB-1 is a transcriptional repressor of HLA-G.

doi: 10.4049/jimmunol.0902053

Figure Lengend Snippet: FIGURE 8. In situ binding of RREB-1 or HDAC1 to the HLA-G pro- moter in a repressive and active-type chromatin. A and B, ChIP performed with JEG-3 (HLA-G ) and M8 (HLA-G ) cells using anti-RREB-1 and anti-HDAC1 Abs on distal and proximal promoter regions (A) Abs target- ing RNA polymerase II (RNApolII), acetylated histone H3 (AcH3), and phosphorylated histone H3 (AcH3 P) on proximal promoter region (B). Immunoprecipitated HLA-G promoter regions are analyzed on agarose gels by semiquantitative HLA-G-specific PCRs targeting proximal and distal HLA-G promoter. Input chromatin (Input) used as PCR control and IgG () are shown. The absence of RREB-1 and HDAC1 binding observed in JEG-3 cells and the absence of RNA polymerase II, acetylated histone H3, and phosphorylated histone H3 binding in M8 cells validate the specificity of Abs used in ChIP assays.

Article Snippet: ChIP assays were performed as previously described (51) using antiRREB-1 from Rockland; anti-acetylated histone H3 (06-599) and antiphosphorylated Ser10 histone H3 (07-081) from Upstate Biotechnology Associates; and anti-RNApolII (C-21) and anti-HDAC1 (H-51) from Santa Cruz Biotechnology.

Techniques: In Situ, Binding Assay, Immunoprecipitation, Control