c-PLA2 Antibody - #AF6329

Figure 6 PLA2 is supposed to be an upstream regulator of ER stress and lipotoxicity. (A) Proposed mechanism by which PLA2 regulates ER stress and hepatocyte death. (B‒C) Determination of cellular PLA2 enzyme activity and LPC levels. (D) Protein levels of the PLA2 and JNK pathways. (E) Immunofluorescence analysis of protein PLA2. Scale bar, 50 μm. (F–G) Time-dependent effects of HN-001 in suppressing PLA2 activity (F) and the PLA2/JNK axis (G). (H–M) Hepatocytes were treated with the PLA2 inhibitor MAFP (3 μmol/L) for 24 h in the presence or absence of PA, and then cells were harvested and subjected to the indicated examinations. (H) Cellular PLA2 enzyme activity. (I) Cellular LPC level. (J) mRNA level of Xbp-1s. (K) ROS level. (L) Caspase 3 activity determination. (M) Viable cell counting. n = 5 independent experiments. (N) Molecular docking analysis between HN-001 and the protein PLA2. The crystal structure of human PLA2 (PDB ID: 1BCI) was selected. The hydrogen bond and hydrophobic force are displayed as blue and yellow dashed lines, respectively. The image was generated in PyMOL. (O) Determination of the binding affinity of HN-001 for the protein PLA2. (P) PLA2 enzyme activity determination. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, compared with blank group (PA-untreated) cells; #P < 0.05, ##P < 0.01, ###P < 0.001, compared with PA-treated cells.

Fig. 4. ROS generation and phosphorylation of VASP, ERK1/2, p38 MAPK, ERK5, JNK, AKT and c-PLA2. (A) Western blot analysis of the expression of NOX2, p67phox, NOX1, NOXO1 and Rac in WT and p47phox-/- platelets. (B) ROS generation in platelets after stimulation with CRP (2 μg/ml) or thrombin (0.5 U/ml) was expressed as mean fluorescent intensity (MFI) (mean ± SE, n = 6) (Student t-test). (C) The phosphorylation level of VASP, ERK1/2, p38, ERK5 and JNK in CRPstimulated platelets was detected by western blot and (D) quantified as a ratio relative to the total level (mean ± SD, n = 3) (Two-way ANOVA). (E) The phosphorylation level of AKT and c-PLA2 was also detected and (F) quantified (mean ± SD, n = 3) (Two-way ANOVA). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3 Change in protein expression of PLA2 in the thick ascending limb during diabetes. **P<0.01 vs. control group; #P<0.05 vs. diabetic group. TNFR:Fc, TNF receptor fusion protein; PLA2, phospholipase A2.

Fig. 3 Effects of ETR and PLA2 antagonists on NOX4 (A, B & D) and pcPLA2 (C) expression in beating rat atria under normoxic conditions. Data were expressed as the mean ± standard error of the mean (n = 5). Cont, control; ET, ET-1; Var, varespladib, an antagonist of sPLA2; CAY, CAY10650, an antagonist of cPLA2. *p < 0.05 vs. control; &p < 0.05 vs. ET-1.