Ab54353

Protein levels were determined using standard laboratory western blot protocols. The primary antibodies used were: anti-CPSF6 (Proteintech 15489-1-AP), anti-CypA (Thermo Scientific PA1-025), anti-TNPO3 (Abcam ab54353), anti-RanBP2 (Abcam ab2938), anti-tubulin (Sigma-Aldrich T6074). Protein levels of Nup153 were determined using anti-nuclear pore complex proteins antibody (a gift from the Blobel laboratory). The secondary HRP-conjugated antibodies used were anti-rabbit (Invitrogen 65-6120) and anti-mouse (Santa Cruz sc-2005). Western blotting of capsid-binding assays was performed by using fluorescent secondary antibodies and signals were quantitated with a LI-COR Odyssey scanner.

Cell lines were cultured in Dulbecco’s Modified Eagle’s Media (DMEM) (Gibco/BRL; Paisley, UK) supplemented with 10% (v/v) fetal calf serum (FCS) and penicillin (Pen; 100 U/ml), streptomycin (Strep; 100 μg/ml) unless otherwise stated. G418 was used at 1000 μg/ml. Puromycin was used at 5 μg/ml. 293 cell lines were incubated at 37°C, 5% (v/v) CO₂. All other cell lines were incubated at 37°C, 10% (v/v) CO₂. Inducible MxB cell lines were made by transduction of HEK293, THP-1 and A3.01 with pLVX-Tet3G (Clontech), containing the Tet-responsive transactivator, and cells were selected for one week in G418. Cells were subsequently transduced with pLVX-mCherryKilled-MxB (modified from Clontech) and selected for one week in puromycin (2 μg/ml). Single cell clones were produced by limiting dilution in 96 well plates and assayed for optimal MxB induction. MxB inducible cell lines were induced for MxB expression by addition of 2 μg/ml of doxycycline for 48 hr prior to infection. Cell lines used in this study HEK293, U87-MG, HeLa, THP-1, A3.01. Cells routinely tested negative for mycoplasma using the Lonza MycoAlert mycoplasma detection kit. Antibodies used in this study; TNPO3 (ab54353, Abcam), CPSF6 (ab175237, Abcam), MxB (sc47197 N-17, Santa Cruz Biotechnology), CypA (BML-SA296-0100, ENZO), Nup358 (C288), Tubulin (DM1A, EMD Millipore), β-actin (ab6276, Abcam).