Anti hnrnp h

Immunoblotting was performed as previously described (Mineo et al., 2016 (link)). The following antibodies were used: anti-PD-L1, anti-IDO, anti-JAK2, anti-STAT1, anti-phospho-STAT1 and anti-β-Actin (13684, 86630, 3230, 9172, 9167 and 3700, respectively, Cell Signaling Technology); anti-hnRNP-H (A300–511A, Bethyl Laboratories).

UV-crosslink RNA immunoprecipitation assay was performed as previously described with some modifications (Mineo et al., 2016 (link)). Briefly, cells were UV irradiated at 400 mJ/cm2 and nuclear extracts were prepared by incubating cells in RLN Buffer (50 mM Tris, 1.5 mM MgCl2, 150 mM NaCl, 0.5% NP-40, protease inhibitors) for 5 min. Nuclei were pelleted by centrifuging at 1,450 × g for 2 min and lysed for 10 min in CLIP Buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.1% Sodium Deoxycholate, phosphatase and protease inhibitors, 100 U/ml RNase inhibitor [New England BioLabs]). Samples were sonicated with microtip, 5 watts power (25% duty) for 60 seconds total in pulses of 1 second on followed by 3 seconds off. DNA was digested incubating samples for 15 min at 37°C in 1X DNase salt solution (2.5 mM MgCl2, 0.5 mM CaCl2) with 30 U TurboDNase. EDTA was added to the samples to a final concentration of 4 mM and samples centrifuged at 16,000 × g for 10 min. Nuclear extracts were precleared with Protein A/G Plus Agarose beads (Thermo Fisher Scientific) and incubated with primary antibody (anti-hnRNP-H) or rabbit IgG control (Bethyl Laboratories) overnight at 4°C. Protein/RNA complexes were precipitated using Protein A/G Plus Agarose beads (Thermo Fisher Scientific). Beads were washed and incubated with Proteinase K (Thermo Fisher Scientific) and RNA was extracted using TRIzol.