Raw WB membrane images for Figure S3 of the paper "Inferring upstream regulatory genes of FOXP3 in human regulatory T cells"
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In Supplementary Figure 3, we analyzed the effect of knocking down NRBF2 on protein expression of FOXP3 in human primary Tregs with Western blotting (WB) approaches. We here provided the original unprocessed WB membrane Tiff images. Although the manuscript includes both computational and experimental arms, we here only listed the authors contributing to the latter.
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Supplementary Figure 3b with Western blotting (WB) results. The samples from left to right follow the order below (after excluding the two boundary lanes with ladders): 1, si_NS 0h, 2, si_NS 10h, 3, si_NS 24h, 4, si_NCOA7 0h, 5, si_NCOA7 10h, 6, si_NCOA7 24h, 7, si_NRBF2 0h, 8, si_NRBF2 10h, 9, si_NRBF2 24h. Where si_NS, the primary human Treg cells were first treated with control scrambled non-specific siRNA for 1 day; si_NCOA7, the primary human Treg cells were first treated with NCOA7-specific siRNA for 1 day; si_NRBF2, the primary human Treg cells were first treated with NRBF2-specific siRNA for 1 day. Following siRNA transfection, those Treg cells were then stimulated with anti-CD3/-CD28 ab/human recombinant IL-2 for different indicated periods (h). We stained with anti-human FOXP3, NRBF2 and GAPDH antibodies, respectively and each of the three raw unprocessed Tiff WB membrane images are attached. We also attached one ppt file to annotate the Tiff images. For more details, please refer to Supplementary Figure 3b of the manuscript and the Methods section. Of note, for the membrane that detected FOXP3, only the upper part of the image presented the samples relevant to Supplementary Figure 3b of our work. It is important to highlight that the WB membranes have already been cut (at ~37Kda and 75Kda for the current analysis) before imaging FOXP3 and NRBF2 bands. Because the size of the GAPDH band is too close to 37Kda, to avoid signal loss, we put the cut membrane parts back before GAPDH imaging. Of note, the visualization mode of the ladder was different from that of the NRBF2 and FOXP3 band imaging. After being visualized separately, the ladder image and band image were then merged. But the ladder was visualized together with the GAPDH band imaging using the same mode. This partially explained why some ladder bands appeared as white (“negative”) in the GAPDH picture (for details, refer to the ladder band size annotation ppt file). The ladder bands appeared normal in the NRBF2 and FOXP3 staining membrane Tiff images.