G box mini

Tumor lysate was generated from 4175/NSG tumors as above for 4 individual mouse tumors per group (DMSO versus TTP488). Western blotting was performed using Invitrogen NuPAGE system as previously described^{13 (link)}. Representative images are shown from each group. Primary antibodies (and dilutions) used were as follows: phospho-Pyk2 (Y402), 1:1000, (Cell Signaling; 3291), total-Pyk2, 1:1000, (Cell Signaling; 3480), phospho-STAT3 (Y705), 1:2000, (Cell Signaling; 9145), total-STAT3, 1:1000, (Cell Signaling; 9139), phospho-Akt, 1:1000, (S473) (Cell Signaling; 4060), total-Akt, 1:1000, (Cell Signaling; 9272), β-actin, 1:4000, (Cell Signaling; 3700). Secondary antibodies used were as follows: Goat Anti-Rabbit IgG H&L (HRP), 1:10000, (Abcam; ab97080), Goat Anti-Mouse IgG - H&L (HRP), 1:10000, (Abcam; ab97040). Western blots were visualized using Pierce ECL Western Blotting Substrate (cat# 32106, ThermoFisher) and a G:Box mini (Syngene).

Samples were lysed in RIPA buffer (20 mM Tris, pH 7.5, 2 mM EDTA, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 1% sodium dodecyl sulphate) supplemented with protease inhibitors (Roche). Proteins were detected using primary and HRP-conjugated secondary antibodies using the SuperSignal West Pico kit (Pierce) visualized on a G:Box mini (Syngene). The following primary antibodies were used: KDM4A (Abcam), β-actin (Sigma), HIF-1α (BD Bioscience), H3 (CST), H3K4me3 (Active Motif), H3K9me3 (Sigma), H3K27me3 (Active Motif) and NDRG1 (CST). All bands were compared against a molecular weight ladder (NEB) and the antibody data sheet, to confirm product-specific detection. Figures presented show all detected bands.

Absolute and relative ZIKV RNA expression levels from the C_t values were quantified using both a standard curve (as previously described [12 (link)]) and the ΔΔC_t method. Relative FcRn mRNA expression was assessed with the ΔΔC_t method. Human or canine GAPDH were the endogenous controls for the ΔΔC_t method for BeWo and MDCK/FcRn cells, respectively. ΔC_t values from MDCK/vector infection experiments were used as the baseline to calculate ΔΔC_t for MDCK/FcRn infection. ΔC_t from cells without infection or without siRNA were used to calculate ΔΔC_t for FcRn expression in infected and siRNA-treated cells, respectively. Differences in RNA expression levels were then calculated using the expression: Fold Change = 2^(−ΔΔCt).
Western blots were imaged using GBOX Mini (Syngene, syngene.com, accessed on 30 November 2022) imaging system and then analyzed with ImageJ (NIH open source, Version 1.53t). The area under the intensity peak curve for the FcRn band was calculated using software tools; this value was then normalized by dividing with the intensity of the corresponding actin band to allow for comparisons across experiments.
Both t tests or one-way ANOVA for column analyses and 2-way ANOVA for grouped analyses (GraphPad Prism 9) were used to assess the differences in expression levels, and p < 0.05 was considered significant.