Fractionation and Western Blot Analysis

Treated cells were harvested in NP40 lysis buffer (1% NP40 + 50 mM Tris-HCl + 150 mM NaCl) and fractionated^{21 (link)}. Briefly, samples underwent two rounds of sonication (60 s, 1 s pulse on/off at 10% power) and centrifugation, prior to re-suspending the insoluble pellet in fresh NP40 lysis buffer. Western blotting used antibodies to detect GFP (Santa Cruz Biotechnology, sc-9996, 1:1000), GAPDH (Santa Cruz Biotechnology, sc-47724, 1:4000), Histone H3 (Cell Signaling Technology, 9715, 1:10,000), hnRNPA1 (ThermoFisher Scientific, PA5-19431, 1:1,000), hnRNPA0 (ThermoFisher Scientific, PA5-57722, 1:1000), and HIS-tag (ThermoFisher Scientific, MA1-21315, 1:5000). α-mouse HRP (ThermoFisher Scientific, A16011, 1:10,000) or α-rabbit HRP (ThermoFisher Scientific, A16023, 1:10,000) were applied prior to detection on the Amerhsam Imager 600 (GE Healthcare Life Sciences). Relative intensities were calculated as the average pixel intensity of heat shocked insoluble fraction bands compared to the average pixel intensity of the cell lysate bands, normalized against background. Intensity measurements were made using ImageJ 1.52a (National Institutes of Health). For each sample at least three independent blots were measured, and data is presented as the mean value of biological replicates +/- standard error of the mean. Statistical significance was measured via Student’s two tailed t-test.