Ab10287

Cells were UV-crosslinked at 4,000 μJxcm² (link) using a Spectrolinker UV Crosslinker, scraped in 200ul of NP40 lysis buffer [50 mM HEPES pH 7.5, 150 mM KCl, 2 mM EDTA, 1 mM NaF, 0.5% NP40, 0.5 mM DTT, complete EDTA-free protease inhibitor cocktail (Roche)], incubated for 15 min at 4°C on a rotating wheel and then centrifuged at 13,000 g for 10 min at 4°C. 2 mg of cellular extract for each sample (IP and IgG) were pre-cleared and 10% of input was collected. Pre-cleared extracts were incubated overnight at 4°C with 2ug of IgG or PDIA3 (anti PDIA3 polyclonal antibody ab10287, Abcam) specific antibodies and then coupled with Dynabeads Protein G magnetic particles at 4°C for 2h on a rotator. After washes with High Salt Wash Buffer (50 mM HEPES-KOH, 500 mM KCl, 0.05% NP40, 0.5 mM DTT, complete EDTA-free protease inhibitor cocktail) beads were resuspended in 200 μl of High Salt Wash Buffer. 50 μl (25%) were kept for protein fraction, resuspended in 30ul of 4× Laemmli sample buffer (Bio-Rad) and 50 mM DTT, incubated 15′ at 70° and analysed by Western blot. The remaining 150ul (75%) were diluted in 75ul of NP40 lysis buffer, treated with Proteinase K and incubated for 30′ at 50°C with shaking. RNA was extracted by miRNeasy Kit (Qiagen), retro-transcribed by Superscript Vilo cDNA synthesis Kit (11754050, Invitrogen) and analyzed by qRT-PCR using primers listed in Table S4.