hnRNP A1 Silencing in HEK 293T Cells

HEK 293T cells were seeded at 1×10⁵ cells per well in 24-well culture plates. Endogenous hnRNP A1 protein silencing was performed over 60% confluent cells using the Lipofectamine 2000 system (#11668019, Invitrogen, Thermo Fisher Scientific Inc.). For hnRNP A1 silencing, a silencer RNA (siRNA) duplex (5′-AATGGGGAACGCTCACGGACT-3′; Integrated DNA Technologies, IDT, Coralville, IA, USA) previously described (23 (link)), targeting the open reading frame (ORF) of the hnRNP A1 mRNA was used at 150 nM for 48h, together with 200 ng of dl HIV-1 IRES plasmid, or at 100, 150 or 250 nM for 48 hr in combination with 200 ng of pNL4.3-RLuc plasmids. For RLuc silencing, 100 nM of a duplex siRNA targeting the RLuc ORF (Renilla siRNA 2, 5′-UAUAAGAACCAUUACCAGAUUUGCCUG-3′, Integrated DNA Technologies) (12 (link),29 (link),32 (link)), was co-transfected with 150 ng of dual-luc plasmids and pSP64 Poly(A) or hnRNP A1-HA plasmids for 48h. The Silencer Select Negative Control #1 siRNA (#4390844, Ambion, Thermo Fisher Scientific Inc.), was used as a non-related scrambled RNA (scRNA) negative control.