Fig 2: Mob2 knockdown increases FlnA phosphorylation at Ser2152 in vitro. (A) C2C12 cells were transiently transfected with EGFP/empty vector control or miRNAs targeting Mob2 (left panel). At 24 h after transfection, cell lysates were prepared and subjected to Western blot analysis using the indicated antibodies. Right panel shows results for the same experiment but performed on age-matched control or patient 1203 fibroblasts. (B) Quantifications representing three biological replicates of (A) summarizing the proportion of phosphorylated Flna at residue 2152 relative to total FLNA and normalized against the loading control Gapdh in C2C12 or age-matched fibroblast controls. Results represented as mean (± SEM). (C) Mean (± SEM) FlnA expression in C2C12 cells expressing control or Mob2 targeting hairpins at the indicated amounts. Expression was normalized against the control using the delta-delta Ct method, as determined by real-time PCR. Expression was normalized against Gapdh and Dimt1 housekeeping genes in the same sample using the relative standard curve method. Mann-Whitney U test; *P < 0.05, **P < 0.005, ***P < 0.001.

Fig 3: ROR2 expression correlates with increases in calpain expression and calpain-mediated cleavage. (A) Western blots comparing expression of downstream targets between a subset of high (n = 6) and low (n = 6) ROR2 expressing RVF patients. (B) Logarithmic regressions comparing ROR2 expression to full length, calpain cleaved, and phosphorylated FLNA (n = 12 total). Y-axis is normalized to the maximal expression of each target protein among these 12 patient samples.

Fig 4: The effects of PN/integrin ß1 on association of Pak1 with FLNA, a substrate for Pak1 kinase activity in VIC lysates. (A,B) VICs were transfected with lentivirus encoding Myc-FLNA for 48 h and then serum starved for 24 h. They were then either untreated or treated with 1 µg/ml of PN for 1 h. (A) Cell lysates were immunoprecipitated without (lane 1, control IgG) or with an antibody against Myc (for FLNA) and immunoblotted with antibodies against Pak1, Myc, or FLNA. (B) VICs were pretreated with cytoskeleton disrupter 2 µM cytochalasin D (CytoD) for 30 min and then treated with 1 µg/ml PN for 1 h (lane 4) and compared with VICs treated with PN alone (lane 3). Cell lysates were immunoprecipitated with an antibody against Myc (for FLNA) and immunoblotted with antibodies against Pak1, Myc, or for FLNA in WCL. Lane 1 is for control IgG. (C) Upper Panel: VICs were cotransfected with the FLNA S2152A mutant, or DN Pak1 containing Pak1 autoinhibitory domain (residues 83–149), or DN Cdc42, or 5 µg/ml of a5 and ß1 integrin blocking antibodies for 2 h. After 48 h, the cells were serum starved, or treated with or without 1 µg/ml of PN as in the experiment in Figure 2G. Pak1 was immunoprecipitated, and a nonradioactive kinase traditional in-gel phosphorylation assay was done using GST fusion proteins of FLNA as substrate. Isotype control IgG was used as control. For details, see the “Materials and Methods” section. Lower Panel: Bars represent densitometric ratios of active phospho pFLNA (S2152)/total FLNA in the lysates. (D) In vitro Pak1 kinase assays from the experiment in panel (C) were further confirmed by assaying using the nonradioactive assay kit (ab138879, Abcam), which is based on the monitoring of ADP formation (see “Materials and Methods” section for details). Data are presented as fold increase in Pak1 kinase activity with respect to control. The data in the experiments in panel (A–D) are representative of =3 independent experiments. The values in experiments (A–B) were determined by densitometry. The values derived from the control cells were designated as 1.0. Error bars are reported as means ± SD of =3 separate experiments.

Fig 5: Proposed RVF model consisting of reactivation of a ROR2 fetal gene program with increasing RVF severity, which results in increased calpain expression and activity leading to calpain-mediated cleavage of cytoskeleton structural proteins including FLNA.