JRFL-SOSIP.R6-IZ-CD40L design and construction. A. Schematic of the SOSIP.R6.L-CD40L design and its various linkers. B. Optimization of the linker between SOSIP.R6 and CD40L using reducing SDS-PAGE analysis of transiently expressed SOSIP.R6-L-CD40L with the different linkers. C. Schematic of the constructs mainly used in this study. D. Reducing SDS-PAGE and Blue Native-PAGE analysis of SOSIP.R6-L3-CD40L and SOSIP.R6-IZ-CD40L proteins secreted from transiently transfected 293T cells. E. Gel filtration analysis of SOSIP.R6-L3-CD40L and SOSIP.R6-IZ-CD40LHis proteins. Concentrated culture supernatants, derived from transiently transfected 293T cells, containing the SOSIP.R6-L3-CD40L or SOSIP.R6-IZ-CD40LHis proteins were fractionated on a Superose-6 column, followed by analysis by SDS-PAGE and western blot. The elution of standard proteins is indicated.