Amicon ultra 15 100 kda centrifugal filters

eIF4B cDNA was amplified from human B cell cDNA and cloned into S-protein/FLAG/streptavidinn binding protein (SFB)-triple tagged destination vector (generous gift from Dr. MS Reddy (CDFD, TG, India) using Gateway cloning system (Invitrogen). USP11 (wt and catalytically inactive (CS)) and USP7 expression constructs were a generous gift from Goedele Maertens & Gordon Peters (Addgene plasmid # 46749). USP11^S453A/D mutants were generated by using site directed mutagenesis kit (Agilent technologies, Palo Alto, CA, USA) according to manufacturer’s recommendations. Expression constructs of USP11 (wt and all mutants) were amplified and cloned into lentiviral-vector system CD513B (System Biosciences, CA). Lentiviral constructs for knockdown of eIF4B, USP11, USP7, and FASN were purchased from Sigma. For lentiviral packaging, HEK293T/17 cells were seeded at 50% confluence and transfected using packing plasmids psPAX2 and pMD2.G (genereous gift from Dr. Didier Trono; Addgene # 12260 and $12259). Lentiviral particles were concentrated using Amicon Ultra-15 100 kDA centrifugal filters (EMD Millipore) per manufacturer’s protocol. DLBCLs were treated with polybrene (4 mg/mL, American Bioanalytical) and centrifuged (35 min, 300×g, 25 °C). Cell pellets were resuspended in virus-containing medium for 48 h, then selected and maintained with puromycin (1 mg/mL).

Expression plasmids for eIF4E1-S209D and eIF4E1-S209A were a gracious gift from Dr Hans-Guido Wendel (Memorial Sloan Kettering Cancer Center, NY). Expression plasmids for eIF4E1, eIF4E3 and eIF4E3-D199 were cloned from previously described constructs7 (link). MNK1, MNK2, MNK1-TD and MNK1-AA were a gracious gift from Dr Herman Gramm (Novartis Institutes for BioMedical Research, Switzerland). All expression plasmids were cloned into a lentiviral-vector system CD513B (System Biosciences, CA). For lentiviral packaging, HEK293T/17 cells were seeded at 40% confluence and transfected the following day with psPAX2 and pMD2.G (Addgene plasmids 12,260 and 12,259; deposited by Dr Didier Trono). Virus-containing medium was concentrated using Amicon Ultra-15 100 kDA centrifugal filters (EMD Millipore) as per the manufacturer’s instruction. HLY-1 and Pfeiffer cells (2x10⁵ cells per ml density in a 10 ml volume) were treated with 4 μg ml⁻¹ of polybrene (American Bioanalytical) and centrifuged for 35 mins at 800 r.c.f. at 30 °C. Cell pellets were resuspended in virus-containing medium for 12 h. Cells were selected and maintained with puromycin (2 μg ml⁻¹).