ERp72 Antibody - #DF12114

Fig. 5. Serine (or cysteine) peptidase inhibitor, clade A, member 3C (Serpina3c) inhibited endoplasmic reticulum overoxidation and its downstream in adipocytes at least partially by suppressing hypoxia-inducible factor 1α (HIF1α)/endoplasmic reticulum oxidoreductase 1α (Ero1α)-protein disulfide isomerase (PDI) signaling. (A) The effect of transcription regulatory factor HIF1α on the promoter activity of protein disulfide isomerase family A member 3 (PDIA3) and PDIA4 in 293T cells was detected by Dual-luciferase reporter assay. (B-H) Serpina3c knockdown (3cKD) 3T3-L1 adipocytes were pretreated with 100 ng/mL recombinant mouse Serpina3c protein (rSerpina3c) or 5 μM HIF1α inhibitor acriflavine (ACF) for 2 hours before being stimulated with 500 μM palmitic acid (PA) for 48 hours. (B) The mRNA levels of indicated genes in 3cKD 3T3-L1 adipocytes. (C, D) The protein levels of indicated genes in 3cKD 3T3-L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (E) Hydrogen peroxide (H2O2) level in 3cKD 3T3-L1 adipocytes was determined. (F, G) The protein levels of mitogen-activated protein kinase (MAPK) signaling pathway in 3cKD 3T3-L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (H) Caspase-3 activity in 3cKD 3T3-L1 adipocytes was determined. (I-L) 3cKD 3T3-L1 adipocytes were pretreated with 10 μM Ero1α inhibitor EN460 or 0.4 μM PDI inhibitor propynoic acid carbamoyl methyl amide 31 (PACMA31) alone or in combination for 2 hours before being stimulated with 500 μM PA for 48 hours. (I) The mRNA levels of indicated genes in 3T3-L1 adipocytes. (J) H2O2 level in 3cKD 3T3-L1 adipocytes was measured. (K) The protein levels of MAPK signaling pathway in 3cKD 3T3-L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (L) Caspase-3 activity in 3cKD 3T3-L1 adipocytes was determined. Data were presented as mean±standard error of the mean. MCS, multiple cloning site; 6xHis, hexahistidine; IL-6, interleukin-6; CCL2 or CCL5, C-C motif chemokine ligand 2 or 5; CXLC1 or CXCL10, C-XC motif chemokine ligand 1 or 10; GRP78, glucose regulated protein 78; CHOP, C/EBP homologous protein; p-JNK, phosphorylated c-Jun N-terminal kinase; p-ERK, phosphorylated extracellular signal-regulated kinase; NS, no significance.