Igf2bp1 antibody

2‐day LIF‐ or RA‐treated mouse ESCs were UV‐crosslinked at 450 mJ/cm². Cell extracts were made using a lysis buffer (100 mM Tris pH 7.4, 150 mM NaCl, 1% NP40, RNase inhibitor, protease inhibitor cocktail, and 1 mM DTT). 1 mg of cell extract was taken for immunoprecipitation following which extracts were pre‐cleared with 50 μl protein A/G resin (Thermo Scientific, 53132) for an hour at 4°C. Pre‐cleared lysates were incubated with Igf2bp1 antibody (Cell Signaling Technologies, 2852S) or IgG (Cell Signaling Technologies, 3900S) control overnight at 4°C. Antibody was immunoprecipitated using protein A/G‐coated resin and subsequently washed four times with lysis buffer. To release the RNA from the protein complex, the pulldown sample was treated with 5 mg/ml proteinase K at 37°C for 10 min. The RNA was extracted using TRIzol. Small RNA libraries were made using the isolated RNA as described using TruSeq Small RNA Prep Kit. The libraries were sequenced and analyzed as discussed previously. For qPCR analysis of target genes, 1 μl of the suspended RNA was reverse‐transcribed with SuperScript III RT (Invitrogen) using random hexamers or tsRNA‐specific RT primer. qPCR of the reverse‐transcribed cDNA was carried out on Bio‐Rad CFX384 Touch machine using Fermentas Power SYBR Green Master Mix and respective gene‐specific or tsRNA‐specific primers.

Total protein extracts were vortex and prepared in lysis buffer (1% Nonidet P-40, 50 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA, 10% glycerol, protease and phosphatase inhibitors). The samples were separated on 12% polyacrylamide denaturing gels and transferred to PVDF membranes. Membranes were saturated for 1 h in 5% milk with 0.1% Tween 20-PBS (PBS-T) solution before incubating with the following antibodies: LIN28B antibody, IGF2BP-1 antibody, Cyclin B1 antibody, p21waf1 antibody from Cell Signaling Technologies (Danvers, MA) and Cyclin D1 antibody, beta-actin HRP conjugated antibody from Santa Cruz Biotechnology (Santa Cruz, CA).