Fig. 5: RASSF2 is completely dependent on stabilization by Hippo kinases in vitro and in vivo.

a Western blot showing time-course of protein expression following addition of 25 μM cycloheximide (CHX) from HEK293T whole cell lysates beginning 48 h post-transfection with RASSF2 expression vector. Quantification of three experiments is shown below. b Western blot showing time-course of protein expression following addition of 25 μM cycloheximide (CHX) from HEK293T whole-cell lysates beginning 48 h post-transfection with RASSF2 expression vector, with (bottom) or without (top) the addition of 10 μM Lactacystin. Data are representative of three experiments. c Schematic of MST1 protein variants expressed in experiments d–f. K59R, substitution of arginine for lysine at amino acid residue 59 results in kinase-dead protein that functions as dominant negative; AID, auto-inhibitory domain; SARAH, Salvador-Rassf-Hippo domain. Red arrows represent endogenous caspase-cleavage sites. d Western blot showing protein from whole-cell lysates in the SKNO-1 t(8;21) cell line transduced with MST1-variant retroviral expression vectors as indicated. c-MST1 caspase-cleaved MST1. e Western blot showing protein from HEK293T whole-cell lysates 72 h post-transfection with indicated expression vectors, with or without the addition of 25 μM cycloheximide (CHX) for the final 8 h as indicated. Data are representative of three experiments. f Quantification of relative change in RASSF2 protein abundance with or without cycloheximide treatment from three experiments described in e. g Western blot showing protein from whole-cell lysates of Lineage-marker negative (Lin−) primary murine hematopoietic cells derived from bone marrow of mice of indicated genotypes. Cells from three mice per genotype are pooled for analysis.