Fig 1: ECH protected PC12 from 6-OHDA-induced ERS through promoting seipin ubiquitination and degradation. (A) ECH down-regulated seipin and ERS-associated protein expression induced by 6-OHDA. Seipin, GRP94, Bip, ATF-4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions (n = 3, 40 µg protein each lane). (B) Knocking down seipin by siRNA attenuated ERS-associated protein accumulation induced by 6-OHDA. Seipin, GRP94, Bip, ATF-4, CHOP, GAPDH protein expression levels detected by Western blotting in groups treated with different conditions (n = 3, 40 µg protein each lane). SiRNA, 50 nM each group transfected and collected to obtain protein lysis after 48 hrs. (C) The relative mRNA levels of seipin were determined using RT-PCR. The results of RT-PCR were normalized to GAPDH and expressed as fold change to control. SiRNA of seipin dramatically inhibited mRNA levels of seipin (P < 0.01), but there was no significant difference among each group transfected by Si-control (n = 3, the values represent the mean ± S.D. of triplicate experiments). (D) ECH promoted seipin degradation under 6-OHDA treatment. MG132, 50 µM; lane1-lane4, 0, 1, 2, 4 hrs, n = 3, 40 µg protein each lane. (E) 6-OHDA blocked seipin ubiquitination but ECH rescued it. Protein lysis of each group was purified and pulled down by anti-seipin antibody and detected by anti-ubiquitin antibody. Ponceau S staining of the whole PVDF membrane containing all samples taken as loading control.
Fig 2: Sig-1R and GRP78 protein expression on the ER after CIRI. (A) The protein levels of Sig-1R and GRP78 were determined by Western blotting. (B) Sig-1R protein levels with increasing reperfusion times. (C) GRP78 protein levels with increasing reperfusion times. The results were normalized to the percentage of Calnexin expression. Data are shown as the mean ± SEM, n = 6 per group. #P < 0.05, ##P < 0.01 compared with the MCAO 0-hour group.
Fig 3: Expression of GRP78 in six PDAC cell lines.Cropped blots/gels were used in the figure and the gels had been run under the same experimental conditions; the full-length blots/gels are presented in Supplementary Figure S3B.
Fig 4: Effect of IT-139 on expression of chaperone protein family(A) Relative levels of GRP78, GRP94, CRT and HSP70 in melanoma SK-MEL-28 cells treated with various dosages of IT-139. Protein levels, depicted by a solid line, and mRNA levels, depicted by dashed lines, were measured with or without Tu treatment. (B) Same as (A) except lung cancer A549 cells were examined.
Fig 5: IT-139 molecule and its effects on human cells(A) Chemical structure of IT-139: sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (molecular weight 502.15g/mol and formula C14H12Cl4N4RuNa). (B) Effect of IT-139 and RuCl3 on GRP78 protein in treated HCT116 cells. Relative GRP78 protein levels were measured after 16 hours of treatment by Western blot with ß-actin serving as loading control. The band intensities were quantitated and graphed below. Tg: thapsigargin. (C) HEK-293T cells were treated with the indicated dosages of IT-139 (0–500 µM) for 16 hours and assayed for GRP78 protein levels. (D) HEK-293T stable cell line harboring the -169-luciferase construct was treated with Tg alone or in combination for 16 hours with 200 µM IT-139 as indicated and assayed for luciferase activity. (E) Relative GRP78 protein levels in primary astrocytes after 200 µM IT-139 treatment alone or in combination with Tg. (F) Relative GRP78 protein levels in HUVECS after 200 µM IT-139 treatment alone or in combination with Tg.
Supplier Page from Abcam for Anti-GRP78 BiP antibody [EPR4040(2)]