Fig 2: The concentrations of SAP, EAT-2 and SHP-2 in NK92 cells.Recombinant SAP, EAT-2, SHP-2 N+C and NK92 cell lysates were analysed by Western blotting using the LI-COR Odyssey Sa system. A representative blot for each protein is shown. 1×106 NK92 cells express 30 ng of SAP and 7 ng of EAT-2. 0.1×106 NK92 cells express 92 ng of SHP-2. One representative blot of each protein is shown.

Fig 3: Depletion of Gas6 induces activation of the PTPN11 and MTOR pathways. (A) Typical expression pattern of Gas6 downstream target genes in GV and MII oocytes. For the PCR, a single oocyte-equivalent mRNA was used as a template for the amplification of each gene. GV and MII oocytes were harvested after in vitro culture for 0 and 16 hours, respectively. Expression levels were calculated from CT values after normalization with H1foo. The asterisks represent statistical significance at p < 0.05. (B) The qPCR analysis of Gas6 downstream target genes in Gas6-silenced MII oocytes, including Ptpn11, Bnip3 and Mtor. Expression levels were calculated from CT values after normalization with H1foo. Control, GFP dsRNA-injected MII oocyte; Gas6 RNAi, Gas6 dsRNA-injected MII oocyte. The asterisks represent statistical significance at p < 0.05. (C) Western blot analysis of p-PTPN11, PTPN11, BNIP3, p-MTOR and MTOR in Gas6-depleted MII oocytes. After Gas6 RNAi, expression levels of p-PTPN11, PTPN11, p-MTOR and MTOR were increased, whereas the expression of BNIP3 was decreased. A protein lysate from 200 MII oocytes was loaded into each lane. a-TUBULIN was used as a loading control.

Fig 4: Validation of RNA-Seq and examination of the abundance of transcripts of mitochondria-related genes by qPCR. (A) Based on the RNA-Seq analysis, among the transcripts of differentially expressed genes, six genes that were involved in mitochondrial proteins (e.g., Phyhipl and Tomm7), downstream target of Gas6 (Ptpn11) and mitophagy (e.g., Atg2b, Binp3 and Esr2) were selected. (B) Changes in the expression of selected genes in Gas6 dsRNA-injected MII oocytes were measured by qPCR. The experiments were performed in biological triplicates of pools of 20 oocytes, and the data are expressed as the mean ± SEM. Expression levels were calculated from CT values after normalization with H1foo. Control, GFP dsRNA-injected MII oocyte; Gas6 RNAi, Gas6 dsRNA-injected MII oocyte. The asterisks represent statistical significance at p < 0.05.

Fig 5: The phosphatase SHP-2 binds NEPH1.A, immunoprecipitation of endogenous NEPH1 from podocytes followed by mass spectrometry analysis identified SHP-2 (N6) as a novel NEPH1 interacting protein. The plot shows the normalized spectrum values of four different bands excised between 75 and 50 kDa (highlighted as C6 and C7 from IgG controls and N6 and N7 from NEPH1 antibody) derived by using Scaffold software (Proteome Software, Inc). B, direct binding was evaluated by mixing purified phosphorylated GST-NEPH1-Cytoplasmic Domain (NEPH1-CD, produced in TKB1 cells) with purified recombinant SHP-2, which showed increased binding of SHP-2 with phosphorylated GST-NEPH1-CD. C, NEPH1 was coexpressed with SHP-2 or the substrate trapping SHP-2 mutant (DM SHP-2) with or without the phosphorylating agent FYN. To evaluate NEPH1 binding, SHP-2 was immunoprecipitated from the cell lysate and Western blotted (WB) with NEPH1 antibody. This demonstrated that NEPH1 bound SHP-2 in the presence of FYN. Significantly enhanced binding was observed with DM SHP-2 indicating that NEPH1 is a SHP-2 substrate. D, control podocytes and podocytes with stable SHP-2 knockdown were treated with growth factors (TNF-a, HGF, and VEGF), and the phosphorylation of endogenous NEPH1 was evaluated using phospho-NEPH1 antibody. NEPH1 phosphorylation was significantly increased only following HGF stimulation of SHP-2 KD podocytes. Experiments B–D were performed in triplicate, repeated three times with similar results, and representative images of the results are presented in the figure. Data are presented as mean ± SEM, and p-values were calculated using the Sidak's multiple comparisons test (two-way ANOVA). **p = 0.01, ***p = 0.001, ****p = 0.0001. SCR, scrambled.