Pk pf647 1

Annexin V was prepared following a published protocol^{28 (link)}, and coupled to either fluoresceinisothiocyanate (Sigma; F7250) or to PromoFluor 647 using its N-hydroxysuccinimidyl ester (PromoCell, Heidelberg, Germany; PK-PF647-1). Drugs used in this study were obtained from Sigma-Aldrich (melphalan; M2011), Selleck Chemicals, München, Germany (bortezomib; S1013, carfilzomib; S2853, nutlin-3A; S8059), and Merck, Darmstadt, Germany (doxorubicin; 324380). A solution of Na-resazurin (Sigma; R7017) in PBS was used as reagent in viability assays (alamarBlue).

Annexin V was prepared according to the protocol detailed in [28] (link), coupled to PromoFluor 647 using its commercially available N-hydroxysuccinimidyl ester (PromoCell, Heidelberg, Germany; PK-PF647-1), and the final concentration adjusted such that 1 µl produced a maximal signal shift in FACS measuremants of MM cells. Stealth siRNA against enhanced green fluorescent protein (cat. no. 12935-145) and custom-built stealth siRNA against human ERK2 (5′-GAGGAUUGAAGUAGAACAGGCUCUG-3′, equivalent to bases 900 to 924 of human ERK2) were obtained from Life Technologies (Darmstadt, Germany). 6-carboxyfluorescein (6-FAM)-labelled siRNA against ERK2 (5′-fluorescein-AAGAGGAUUGAAGUAGAACAG-dTdT-3′, a shorter version of the sequence mentioned above) was from Qiagen (Hilden, Germany). The pcDNA3.1-CD4Δ and the pSUPER-ERK2 vectors are described in [21] (link).