α mouse 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).

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.

Protein extracts used for Western Blot were generated by transfecting 5 × 10⁶ HeLa cells with 10 µg of expression vector DNA and lysing cells with RIPA buffer after 48 hr. Lysates were passed through a 23-gauge needle, incubated 30 min on ice, then centrifuged at 10000 g and 4°C for 10 min to remove cell debris. Total protein concentrations were determined via Bradford assay (Pierce Coomassie Plus [Bradford] Assay Kit, Thermo Fisher). Proteins were separated by discontinuous SDS-PAGE and transferred onto nitrocellulose membranes (1 hr at 100 V). Membranes were stained with α-SB antibody (RRID:AB_622119, R and D Systems, 1:500, 2 hr) and α-goat-HRP (RRID:AB_258425, Sigma, 1:10000, 1 hr) or with α-actin (RRID:AB_2223496, Thermo Scientific, 1:5000, 2 hr) and α-mouse-HRP (RRID:AB_228313, Thermo Scientific, 1:10000, 1 hr) for the loading control. Membranes were visualized using ECL Prime Western Blotting reagents.