Fig 2: AKT activity is not required for neutrophil survival. (A, B) Neutrophils were treated with MK-2206 for 30 minutes (A) or Afuresertib for 60 minutes (B) prior to further incubation in the presence or absence of LVS. Apoptosis was assessed at 4, 10, 18, and 24 hours using Annexin V-FITC/PI staining and flow cytometry. Data are the mean ± SEM of three independent experiments. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001, as indicated. (C, D) Immunoblots of cell lysates were probed to detect phosphorylated AKT (P-AKT) with ß-actin as the loading control. (C) Extent of AKT phosphorylation at 0, 3, 6, 12 and 24 hours in uninfected and infected cells, as indicated. (D) Effects of Afuresertib and MK-2206 on AKT phosphorylation in LVS-infected cells. Data shown are representative of three determinations.

Fig 3: Effects of SG-SP1 on activation of NF-?B, and signaling proteins in mast cells. RBL-2H3 were sensitized with anti-DNP IgE. After incubation overnight, the cells were pretreated with or without SG-SP1 and GA for 1 h and then challenged with DNP-HSA. Activation of NF-?B and signaling proteins were assayed by Western blot (N-: nuclear, p-: phosphorylated). Lamin B1, ß-actin, and total form of Lyn, Fyn, Syk, and Akt were used as loading controls. The band intensity was quantified and presented as the means ± SEM of three independent experiments. *, **, and *** indicate statistically significant differences from the DNP-HSA-challenged group at a value of p < 0.05, 0.01, and 0.001, respectively.

Fig 4: E-peptides affect IGF-IR downstream signaling. A–C. C2C12 cells were treated as in Figure 2, with 0 nM or 2 nM IGF-I and optimal doses of the E-peptides (EA 1 µM, EB 10 nM) for 20 minutes. B–C. Quantification of Akt and ERK1/2 phosphorylation after EA (B) or EB (C) treatment. EA and EB alone are compared to NoTx, while IGF-I with E-peptides are compared to 2 nM IGF-I alone. Data are presented as the effect on phosphorylation after E-peptide treatment compared to NoTx or NoPeptide plus IGF-I. Bars represent means ± s.e.m. of N = 3–4 replicates. †, p<0.05 for comparisons of 0 nM IGF samples to their 2 nM IGF-I counterparts via student t-tests.

Fig 5: Microscopical assay to detect internalized PDGFA) Schematic of bt-PDGF detection. Cells are stimulated with bt-PDGF 1. Following internalization, biotins on extracellular bt-PDGF are removed by a reducing agent 2 and only intracellular bt-PDGF is detected by anti-biotin antibodies 3. B) Images of cells stimulated with 100 ng/mL bt-PDGF: before (left) and after (right) removal of extracellular biotins. Scale bar 20 µm. C) Activation of PDGFR and its downstream effectors upon stimulation of cells with 100 ng/mL unlabeled PDGF or bt-PDGF, visualized by immunoblotting with phospho-specific antibodies against tyrosine (pTYR; for PDGFR phosphorylation), STAT3, AKT and ERK.