Fig 1: Robust expression of Plexin-B2 ligands. Immunoblot analyses of protein lysates of rhabdomyosarcoma cell lines, using the known ligands of Plexin-B2, i.e., SEMA4C, SEMA4D, and SEMA4F antibodies in panels (a), (b), and (c), respectively. SEMA4C is uniformly expressed between eRMS and aRMS cell lines. SEMA4D protein expression is increased in eRMS cell lines compared to aRMS cell lines. In contrast, SEMA4F protein expression is increased in aRMS cell lines compared to eRMS cell lines. GAPDH and ß-actin are used as loading controls.
Fig 2: Structural molecular modeling of PlxnB2 wild-type and G842C-mutated form A, D, E Most representative conformations of IPT3 domains of PlxnB2 p.G842C (A, in red), PlxnB2 p.G842C 842–845 (D, in blue) and PlxnB2 p.G842C 842–860 (E, in green) compared with WT most representative conformation (in gray).B, C, F, G RMSD plots of the concatenated replicas of the 4 mentioned IPT3 domains [PlxnB2 WT, p.G482C, p.G842C/842–845, and p.G842C/842–860, respectively].
Fig 3: Expression of G842C-PlxnB2 controls EGFR phosphorylation and intracellular signaling Phospho-EGFR analysis by western blotting in control and PlxnB2-silenced AS43 cells. Images show representative results of n = 3 independent experiments, while the graph at the bottom shows the mean ± SD of normalized band quantification. The statistical significance across replicates was verified by unpaired t-test: ****P < 0.0001.Phospho-EGFR analysis by western blotting in AS901 and AS906 cells either mock, or overexpressing WT or G842C-mutated PlxnB2. Images show representative results of n = 3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by one-way ANOVA followed by Tukey test: AS901 PlxnB2-G842C vs. PlxnB2-WT *P = 0.0434, PlxnB2-G842C vs. mock *P = 0.0211; AS906 PlxnB2-G842C vs. PlxnB2-WT *P = 0.0436, PlxnB2-G842C vs. mock *P = 0.0481.Western blotting analysis of pERK and pS6 intracellular signal transducers in the indicated agnospheres, after 6-h treatment with the EGFR inhibitor cetuximab (50 µg/ml). Images show representative results of n = 3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by Sidak's multiple comparison test between cetuximab-treated and untreated conditions, per each group: AS43 *P = 0.0153, ***P = 0.0001, ****P < 0.0001; AS901 *P = 0.0201. Source data are available online for this figure.
Fig 4: Multiple alignment of the primary sequences of IPT domains found in Plexin family members and in the homologous Met receptorIn the first line is reported the G842C-mutated sequence of IPT3 domain of PlexinB2, found in CUP samples. The primary sequences of corresponding IPT3 domains in plexin family members, and in Met receptor tyrosine kinase, are aligned. Nucleotide positions at start/end of the nucleotide stretches are indicated. Highlighted in yellow are highly conserved amino acid residues. Cysteines engaged in disulfide bonds are underlined. The conserved glycine mutated in PlxnB2 is indicated in bold, as well as neighboring cysteines potentially engaged in covalent links.
Fig 5: Effect of Plexin-B2 siRNA knockdown on cell migration. Crystal violet quantification of transwell migration and invasion assays reveals that in Rh30 and Rh41 (aRMS) cell lines, the migration and invasive properties are compromised upon Plexin-B2 knockdown in the context of serum bait. In contrast, migration property is increased in RD (eRMS) cell line. CF-1, a primary cell line, shows minimal effect on migration after Plexin-B2 knockdown. (The data on graphs show means ± SEM. *p < 0.05, ** p < 0.01, *** p < 0.001).
Supplier Page from Abcam for Anti-Plexin B2/MM1 antibody [EPR9965]