loading controls vinculin  (Bio-Rad)

Journal: Breast Cancer Research : BCR

Article Title: Secreted frizzled-related protein 2 monoclonal antibody-mediated IFN-ϒ reprograms tumor-associated macrophages to suppress triple negative breast cancer

doi: 10.1186/s13058-025-02176-6

Figure Lengend Snippet: SRP2 mAb treatment is associated with an increase in IFN-ϒ levels in TAMs and a significant boost in T-cell proliferation. A TAM enriched single cell suspension was treated in vitro for 1 h with 10uM IgG1 control or 10uM hSFRP2 mAb. Cell lysates were collected and analyzed with western blot. IFN-ϒlevels increase in TAMs treated for 1 and 24 h with hSFPR2 mAb. Full Westerns in Supplemental Fig. . The intensity ratio represents the protein levels from test samples compared to the vinculin loading control as described in Material and Methods. B qRT-PCR results show a 2.35-fold increase in IFN-ϒmRNA levels in 1-h hSFRP2 mAb-treated TAMs compared to 1-h IgG1 control-treated TAMs (n = 3 for both groups, * p < .0.002). Graph created with BioRender.com. C Analysis of IFN-ϒand SFRP2 expression TCGA data from 1075 entries showed a statistically significant inverse correlation between IFN-ϒ and SFRP2 ( p < 0.0001). D Flow cytometry analyzing T-cell proliferation after co-culture of TAMs treated with IgG1 control or hSFRP2 mAb showed significant MFI increases in CD25 in T-cells co-cultured with hSFRP2 mAb treated TAMs when compared to T-cells co-cultured with IgG1 control treated TAMs (n = 3, ** p < .05). E Flow cytometry analyzing T-cell proliferation after co-culture of TAMs treated with IgG1 control or hSFRP2 mAb showed significant MFI increases in CD69 in T-cells co-cultured with hSFRP2 mAb-treated TAMs when compared to T-cells co-cultured with IgG1 control-treated TAMs (n = 3, *** p < .05)

Article Snippet: Protein levels from test samples were compared to the vinculin loading control (Cell Signaling Technology, cat # 4650, RRID: AB_10559207).To do this, densities were calculated by multiplying the average intensity by the surface of each band, and the percentage of control was calculated using the following formula: marker of interest total density × 100/loading control total density.

Techniques: Suspension, In Vitro, Control, Western Blot, Quantitative RT-PCR, Expressing, Flow Cytometry, Co-Culture Assay, Cell Culture