Figure 4 . For loading controls, see
Figure 4 , D and E . B , representative levels of pSTAT1(Y701) and total STAT1 are shown for the A549 ouabain and digoxin experiments in
Figure 5 . For loading controls, see
Figure 5 , C and D . The dashed vertical lines indicate one lane (ladder) that has been spliced out. C , quantification of pSTAT1(Y701) in western blots of NTC or ATP1A1 siRNA transfected TNF/IFN-ɣ (24 h) stimulated MDA-MB-231 cells treated with 100 nM ouabain. ∗ p < 0.05, ∗∗∗ p < 0.001, Two-factor ANOVA, Tukey’s multiple comparisons. D , STAT1 activation time-course experiment in MDA-MB-231 cells treated with 100 nM ouabain or 0.05% DMSO and stimulated for 30 min, 2 h, 8 h, or 24 h with TNF/IFN-γ. Mean intensities ±standard deviation, measured by densitometry of blots from three independent time-course experiments, are listed under the pSTAT1(Y701) long and total STAT1 panels. " width="100%" height="100%">
Journal: The Journal of Biological Chemistry
Article Title: Inhibition of the Na + /K + -ATPase by cardiac glycosides suppresses expression of the IDO1 immune checkpoint in cancer cells by reducing STAT1 activation
doi: 10.1016/j.jbc.2022.101707
Figure Lengend Snippet: ATP1A1 knockdown enhances cardiac glycoside-mediated inhibition of STAT1(Y701) phosphorylation. A , representative levels of pSTAT1(Y701) and total STAT1 are shown for the MDA-MB-231 ouabain and digoxin experiments in Figure 4 . For loading controls, see Figure 4 , D and E . B , representative levels of pSTAT1(Y701) and total STAT1 are shown for the A549 ouabain and digoxin experiments in Figure 5 . For loading controls, see Figure 5 , C and D . The dashed vertical lines indicate one lane (ladder) that has been spliced out. C , quantification of pSTAT1(Y701) in western blots of NTC or ATP1A1 siRNA transfected TNF/IFN-ɣ (24 h) stimulated MDA-MB-231 cells treated with 100 nM ouabain. ∗ p < 0.05, ∗∗∗ p < 0.001, Two-factor ANOVA, Tukey’s multiple comparisons. D , STAT1 activation time-course experiment in MDA-MB-231 cells treated with 100 nM ouabain or 0.05% DMSO and stimulated for 30 min, 2 h, 8 h, or 24 h with TNF/IFN-γ. Mean intensities ±standard deviation, measured by densitometry of blots from three independent time-course experiments, are listed under the pSTAT1(Y701) long and total STAT1 panels.
Article Snippet: Membranes were blocked with 2% BSA at least 1 h at room temperature and probed overnight at 4 °C (1:1000) for the following primary antibodies to PD-L1 (E1L3N), STAT1 (9172), pSTAT1 Tyr-701 (D4A7), SOCS3 (2923S), IDO1 (D5J4E) all from Cell Signaling Technology, or ATP1A1 (ab7671 464.6), from Abcam, and for 1 h at room temperature for GAPDH (6C5), from Abcam.
Techniques: Knockdown, Inhibition, Phospho-proteomics, Western Blot, Transfection, Activation Assay, Standard Deviation
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