α rabbit hrp

The following primary antibodies were used for Immunoblotting, ChIP or IF applications: α-JAZF1 (1:500, produced by Pineda, animal 1), α-ZNHIT1 (1:1000, HPA019043, Sigma-Aldrich, Soeborg, Denmark), α-GFP (1:1000, 11814460001, Sigma-Aldrich), α-H2A.Z (1:1000 or 5 µg/IP, 39944, Active motif, Carlsbad, CA, USA), α-H2A.Zac (1:1000 or 1 µg/IP, ABE1363, Merck Millipore, Darmstadt, Germany), α-alpha Tubulin (1:1000, 39527, Active motif), α-H4K5ac (1:1000, 39700, Active motif), α-H4K16ac (1:1000, 39167, Active motif), α-H2AK5ac (1:1000, 39108, Active motif), α-H3K14ac (1:1000, 39599, Active motif), α-H3 (1:1000, ab1791, Abcam, Cambridge, MA, USA), α-JAZF1 (1:100, HPA066967, Atlas Antibodies, Stockholm, Sweden), α-Fibrillarin (1:100, NB300-269, Novus Biologicals, Centennial, CO, USA), α-Coilin (gift from Grahmam Dellaire). The following secondary antibodies were used for immunoblotting or IF applications: α-rabbit-HRP (1:20000, 31460, Thermo Fisher Scientific), α-mouse-HRP (1:20000, 31430, Thermo Fisher Scientific), α-mouse-Alexa Fluor 488 (1:200, A-11017, Thermo Fisher Scientific), α-rabbit-Alexa Fluor 594 (1:200, A-11072, Thermo Fisher Scientific), α-rabbit-Alexa Fluor 488 (1:200, A-11070, Thermo Fisher Scientific), α-mouse-Alexa Fluor 594 (1:200, A-11020, Thermo Fisher Scientific).

HEK293T were transfected with specified plasmids. 17–24 hr post transfection, HEK293T cells were washed with 1 mM CaCl₂/PBS, lifted off the dish with 2 mM EDTA/PBS, pelleted and lysed by incubating in lysis buffer (150 mM NaCl, 25 mM HEPES, 1 mM EDTA, 0.5% NP-40, 1x PhosSTOP phosphatase inhibitor (Roche), 1x HALT protease inhibitor (Thermo Fisher Scientific) for 30 min, and bath sonicated in ice for 3 min. Cell debris was pelleted at 18,000 rcf for 10 min. Cell lysate were precleared with 15 μl of GFP-Trap (Chromotek) for 30 min at 4°C, and incubated with 15 μl of fresh GFP-Trap beads overnight at 4°C. The beads were washed with lysis buffer five times, before boiled in Laemmli sample buffer and separated on 4–20% acrylamide gradient gels by SDS-PAGE. Proteins were transferred onto nitrocellulose membrane and probed with primary antibodies, α-Grb2 (1:5000, Clone 81/Grb2, BD Biosciences), α-tubulin (1:5000, Clone YL1/2, Thermo), α-pTyr (1:2000, Phospho-Tyrosine (P-Tyr-1000) MultiMab Rabbit mAb mix #8954, Cell Signaling Technology), α-GFP (1:10000, Clone 3E6, Life Technologies or 1:5000, A21312, Life Technologies), and secondary antibodies, α-mouse HRP (1:10,000, Upstate Biotechnology or 1:5000, Jackson Labs), α-rabbit HRP (1:5000, 65–6120, Thermo Fisher), α-rat AlexaFluor 647 (1:5000, Life Technologies). Western blots were imaged on ChemiDoc (Bio-Rad).

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.