PDIA3 Antibody - #DF6230

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.

Figure 4. Expression and subcellular localization of PDIA3. (A) mRNA expression profile of PDIA3 in different tissues of dairy cows by quantitative real-time PCR method; (B) subcellular localization of PDIA3 in bovine mammary alveolar cells observed by immunofluorescence assay. PDIA3 (green); endoplasmic reticulum (ER) signals (red); nucleus stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, blue). Scale bar represents 10 μm.

Figure 5. Detection of the efficiency of PDIA3 overexpression and knockdown in bovine mammary alveolar cells. (A) Map of PDIA3 overexpression vector (left), and agarose gel electrophoresis of pCDNA3.1- PDIA3 vector digested with restriction endonucleases (right). M = DNA marker. Lane 1 (L1) shows 2 bands of 2,248 bp and 4,671 bp with Mlu I and Xho I digestion; lane 2 (L2) shows the pCDNA3.1- PDIA3 plasmid. (B) Cells were transfected for 48 h with pCDNA3.1- PDIA3 overexpression vector (OE), pCDNA3.1 empty vector (EV), PDIA3 siRNA (KD), or negative control siRNA (NC), and the relative mRNA levels of PDIA3 were analyzed by quantitative real-time PCR. (C) Protein levels of PDIA3 were determined by western blot analysis, and quantitative results show fold increases in PDIA3/GAPDH ratios. **P < 0.01, compared with control group; ns = not significant. Error bars represent SEM. siRNA = small interfering RNA.