Pcr blunt 2

cDNAs containing the entire coding region of full-length CD28, CD28i, and CD28Δex2, which do not contain nucleotides for the stop codon, were obtained by RT-PCR analysis of Jurkat cells using the specific primer pairs shown in Additional file 6. cDNAs encoding each CD28 splicing isoform were inserted into pCR-blunt II (Thermo-Fisher Scientific) and subcloned into pAcGFP1-Hyg-N1 (Takara-bio) using XhoI.

BiFC analysis was performed in leaf cells of 3–4-week-old N. benthamiana as described in earlier studies⁸ . Coding sequences of XPO1a and XPO1b were first cloned into pCR BLUNT II (Thermo Scientific). DNA fragments were then digested and fused to the full-length eGFP (aa1-241), the N-terminal fragment of eGFP (N-eGFP; aa1-aa155), or the C-terminal fragment of eGFP (C-eGFP; aa156-aa241) and then cloned into pENTR. LR recombination with the pMDC32 destination vector was then performed. Generation of full-length eGFP, N-terminal eGFP, and C-terminal eGFP fused WUS in pMDC32 destination vector was previously described⁸ . Mutations for EAR-like domain were introduced with primers from Supplementary Table 5. Empty N-terminal eGFP and C-terminal GFP were used as negative controls. Agrobacterium tumefaciens GV3101 was transformed with the BiFC constructs and infiltrated into leaves of 3–4-week-old N. benthamiana plants using agroinfiltration buffer solution (10 mM MES pH 5.7, 10 mM MgCl₂, 20 uM Acetosyringone). Infiltration of corresponding BiFC plasmids along with the p19 suppressor of gene silencing plasmid was introduced as a 1:1:1 cocktail. Fluorescence of eGFP was monitored using a Leica SP5 Confocal microscope. eGFP fluorescence was detected with 488 nm excitation and emission was collected between 500–525nm. Plastid autofluorescence emission was collected between 631–700 nm.