Cell transfection and lentiviral transduction

For western blotting in HEK293 cells, 2.6*10⁵ cells were seeded per well in 12-well plates. For co-immunoprecipitation of proteins ectopically expressed in HEK293 cells, 6*10⁵ cells were seeded in 60 mm dishes. The cells were transfected after 24 hr with plasmid DNA using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Lentiviral particles to transduce K562 cells were produced in HEK293T cells using psPAX2 and pMD2.G packaging plasmids as described elsewhere (Barde et al., 2010). For infection, 1*10⁶ K562 cells were grown in 10 ml of virus-containing RPMI-1460 medium for 24 hr. To achieve shRNA-mediated depletion of BMP2K, MISSION shRNA plasmids were used. The empty pLKO.1 plasmid (SHC001), and the construct expressing non-targeting shRNA (SHC202) served as controls. After initial testing (not shown in the manuscript figures) of 5 different shRNA sequences (TRCN0000000914, TRCN0000000915, TRCN0000000916, TRCN0000000917, TRCN0000226438), only one (TRCN0000000915, shBMP2K) was found to efficiently downregulate BMP2K expression as assessed by western blotting. To achieve shRNA-mediated depletion of specific BMP2K splicing variants, shRNA sequences were designed using the siRNA Selection Program (Yuan et al., 2004) and cloned into pLKO.1 – TRC cloning vector. These sequences are listed in Supplementary file 1-Table 3. To achieve CRISPR/Cas9-mediated inactivation of BMP2K gene, four different gRNA sequences (Doench et al., 2016) were cloned into the lentiCRISPRv2 vector and were lentivirally introduced into K562 cells. Their efficiency of gene expression silencing was tested by western blotting and two sequences causing the strongest reduction of BMP2K protein levels were chosen for further experiments. gRNAs used in this study are listed in Supplementary file 1-Table 4. For BMP2K depletion, cells were transduced with lentiviral particles containing control or gene-targeting vectors for 24 hr and selected with 2 µg/ml puromycin for 72 hr.

For overexpression of EGFP-tagged BMP2K variants, K562 cells were transduced for 24 hr with lentiviral particles containing pUltra-EGFP-BMP2K-L or -S plasmids and analyzed after three subsequent days of culture.