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. 2023 Aug 30;10:1187490. doi: 10.3389/fcvm.2023.1187490

Figure 1.

Figure 1

CHK1 regulates L-flow-induced SENP2 S344 phosphorylation, subsequently suppresses ERK5 and p53 SUMOylation: (A) HUVECs transduced with Ad-SENP2 WT or Ad-SENP2 S344A were exposed to L-flow for 0 and 30 min, and the levels of SENP2 (p-S344 and total), ERK5 (p-T-E-Y and total), and actin (loading control) were determined using Wes. An increase in p-ERK5-T-E-Y indicated successful generation of L-flow, in addition to elongated cell shape (50, 52). (B) HUVECs were exposed to D-flow for 0, 20, 40, and 60 min, and the levels of SENP2 (p-S344 and total) were determined using immunoblotting. (C) HUVECs pretreated with 250 nM GDC 0575 were exposed to L-flow for 0, 20, 40, and 60 min, and the levels of SENP2 (p-S344 and total), CHK1 (p-S280, p-S345, and total), and actin were determined using Wes. (D, upper panel) The graphs present quantified data from 3 independent experiments from (C) (n = 3) (*p < 0.05, **p < 0.01, two-way ANOVA). (D, lower panel) The graphs present quantified data from 3 independent experiments from (B) (n = 3). N.S. indicates no significance. (E–G) The graphs present quantified data from 3 independent experiments from (C) (n = 3) (*p < 0.05, **p < 0.01, two-way ANOVA). (H) WT mouse lung ECs (WT MLECs) and SENP2 S344A KI MLECs were exposed to L-flow for 0 and 30 min, and the levels of SENP2 (p-S344 and total) were determined by immunoblotting. (I) WT MLECs and SENP2 S344A KI MLECs were exposed to L-flow for 0 and 30 min. Lysates were immuno-precipitated with the SUMO2/3 antibody (Signal-SeekerTM SUMOylation 2/3 Detection Kit, #BK162; Cytoskeleton, Inc.), and ERK5 or p53 antibody was used to detect SUMOylated ERK5 or p53 using Wes, respectively. The SUMO2/3 antibody was also used to confirm equal immunoprecipitation across the samples. Control beads were used as the negative control. ERK5, SENP2, and p53antibodies were used to detect their expression in total cell lysates using Wes. (J) The graphs present quantified data from 4 (upper panel) and 5 (lower panel) independent experiments (*p < 0.05, two-way ANOVA).