For cloning of the SunTag-FL2 construct, we used the SINAPs plasmid from the Singer lab (Addgene #84561)44 and a tdTomato-FL2 (containing the 3’UTR of FL2) plasmid previously cloned in-house using the tdTomato-C1 vector (Addgene #54653), a human FL2 clone in pANT7_cGST (DNASU, Arizona State University, Tempe, AZ, clone: HsCD00403041) and a human FL2 3’UTR construct (Switchgear Genomics #S811553). The Ubc promoter, flag tag and SunTag reporter were cloned from the SINAPs construct to replace the CMV promoter and tdTomato reporter of the tdTomato-FL2 plasmid using NEBuilder HiFi DNA Assembly technology (NEB).
The plasmids used for IMP RBP overexpression, GFP-ZBP133 (link), GFP-IGF2BP2 and GFP-IGF2BP2 KH3 mutant39 (link), were from the Singer laboratory. The Lin28 plasmids were purchased from Addgene; Lin28A (#51371), Lin28B (#51373) and Lin28A-mCCHC (#51372). The miRNA let-7 inhibitor sequence (3’-CUCCAUCAUCCAACAU-5’) was previously validated for broad let-7 family inhibition (Frost & Olson, 2011). The custom miRCURY LNA miRNA inhibitor was purchased from Qiagen (Cat num 339146).
The plasmids used for IMP RBP overexpression, GFP-ZBP133 (link), GFP-IGF2BP2 and GFP-IGF2BP2 KH3 mutant39 (link), were from the Singer laboratory. The Lin28 plasmids were purchased from Addgene; Lin28A (#51371), Lin28B (#51373) and Lin28A-mCCHC (#51372). The miRNA let-7 inhibitor sequence (3’-CUCCAUCAUCCAACAU-5’) was previously validated for broad let-7 family inhibition (Frost & Olson, 2011). The custom miRCURY LNA miRNA inhibitor was purchased from Qiagen (Cat num 339146).