human recombinant grp78 bip  (StressMarq)

Journal: Molecular Oncology

Article Title: The synthetic oleanane triterpenoid CDDO‐2P‐Im binds GRP78 / BiP to induce unfolded protein response‐mediated apoptosis in myeloma

doi: 10.1002/1878-0261.13447

Figure Lengend Snippet: CDDO‐2P‐Im binds GRP78 and activates the PERK and IRE1α branches of the UPR in a dose‐dependent manner. (A) Cell lysate of RPMI‐8226 was incubated with DMSO or CDDO‐2P‐Im for 30 min. Lysates were then subjected to 0.025 mg·mL −1 pronase for 30 min at 4 °C, followed by SDS/PAGE and western blot using antibodies against GRP78 and GAPDH. CDDO‐2P‐Im protects GRP78 from pronase cleavage compared with DMSO control. (B) Thermal shift assay was performed on recombinant GRP78 treated with control (DMSO) or CDDO‐2P‐Im. Relative fluorescent units (RFU) were measured by a SpectraMax Paradigm plate reader. The melting temperature (Tm) has a shift of −1.9 °C with the treatment of CDDO‐2P‐Im. (C, D) ARH‐77 and RPMI‐8226 cells were incubated with CDDO‐2P‐Im for 6 h. Cells were lysed and samples were prepared for SDS/PAGE. Western blot of UPR signaling proteins and downstream targets were performed. (E) 5T33 tumors were extracted from mice 15 days after tumor injection. Mice were treated once with either vehicle or 24 mg·kg −1 CDDO‐2P‐Im for 12 h prior to extraction. Samples were immunoblotted for UPR proteins. (F) RNA‐Sequencing data for ATF6 regulated genes showed no differences between Control, 0.1 μ m CDDO‐2P‐Im, and 0.4 μ m CDDO‐2P‐Im in RPMI‐8226 cells. (G, H) Western blot of ATF6 in ARH‐77 and RPMI‐8226 show no changes at 6 h with the treatment of CDDO‐2P‐Im. Values were given as mean ± SD. Fragments Per Kilobase of transcript per Million mapped reads is abbreviated as FPKM. Western blot and DARTS data are representatives of at least three independent experiments for ARH‐77 cells and RPMI‐8226 cells. Thermal shift assay was performed three independent times and representative data was shown. Western blot of 5T33 tumors was performed once. RNA‐Sequencing was performed once. Student t ‐tests were performed to calculate P value. 2P‐Im, CDDO‐2P‐Im; CDDO, CDDO‐2P‐Im.

Article Snippet: 10 μ m of recombinant GRP78 protein (PROTP11021; Boster Biological Technology, Pleasanton, CA, USA) was incubated with 150 μ m 2P‐Im controlled with 1% DMSO, and thermal shift assay was performed as previously described [ ].

Techniques: Incubation, SDS Page, Western Blot, Control, Thermal Shift Assay, Recombinant, Injection, Extraction, RNA Sequencing

Journal: Molecular Oncology

Article Title: The synthetic oleanane triterpenoid CDDO‐2P‐Im binds GRP78 / BiP to induce unfolded protein response‐mediated apoptosis in myeloma

doi: 10.1002/1878-0261.13447

Figure Lengend Snippet: Inhibition of the PERK‐ATF4‐CHOP arm of the UPR partially rescues CDDO‐2P‐Im‐induced apoptosis. (A) Cell lysate of WT and PERK KO RPMI‐8226 were probed for PERK protein by western blot. (B) WT and DDIT3 (CHOP) KO RPMI‐8226 were incubated with DMSO or CDDO‐2P‐Im for 6 h and lysed for protein analysis. Western blot of CHOP was performed to investigate the knockout of CHOP protein. (C) Cells were preincubated with DMSO or CDDO‐2P‐Im for 24 h and evaluated for cell viability by CellTiter‐Glo™. (D) Cells were preincubated with DMSO or 0.25 μ m ISRIB for 3 h before treating with DMSO or CDDO‐2P‐Im for an additional 16 h. Cells were then measured for cell viability by CellTiter‐Glo™. Controls were cells that were not treated with CDDO‐2P‐im but were treated with DMSO or ISRIB where appropriate. (E–H) PERK KO and WT RPMI‐8226 cells or ARH‐77 cells pretreated with ISRIB for 3 h were incubated with DMSO control or 0.4 μ m CDDO‐2P‐Im for 6 h. (E, F) Cells were extracted for RNA and qRT‐PCR was performed to investigate changes in the UPR. (G, H) In another round of experiments, cells were also extracted for protein to confirm such changes in the UPR. Values were given as mean ± SD. Student t ‐tests were performed to calculate P value. * P < 0.05 and ** P < 0.01, compared with control. Cell viability data are representative of three independent experiments. Western blots and qRT‐PCR data are representatives of two independent experiments. (I) We provide a working model of the actions of CDDO‐2P‐Im in cancer cells. At low concentrations (panel A), 2P‐Im binds and inhibits KEAP1, an adaptor protein for ubiquitin ligase that negatively regulates Nrf2 levels. Nrf2 will translocate to the nucleus and dimerize with small Maf proteins (sMaf) to activate the transcription of Nrf2 target genes. At higher concentrations (panel B), 2P‐Im binds GRP78/BiP, resulting in the activation of the UPR. GRP78 dissociates from its binding partners leading to the phosphorylation of PERK and IRE1α and activation of these branches of the UPR. The transcription factors, CHOP and XBP1, will cause UPR‐associated gene expression changes which when prolonged will lead to apoptosis. Interestingly, XBP1 is known to increase the expression of HRD1, a negative regulator of Nrf2, and independent of KEAP1. 2P/2P‐Im, CDDO‐2P‐Im; ISRIB, integrated stress response inhibitor; WT, wild‐type; KO, knockout.

Article Snippet: 10 μ m of recombinant GRP78 protein (PROTP11021; Boster Biological Technology, Pleasanton, CA, USA) was incubated with 150 μ m 2P‐Im controlled with 1% DMSO, and thermal shift assay was performed as previously described [ ].

Techniques: Inhibition, Western Blot, Incubation, Knock-Out, Control, Quantitative RT-PCR, Ubiquitin Proteomics, Activation Assay, Binding Assay, Phospho-proteomics, Gene Expression, Expressing

Journal: Microbial Cell Factories

Article Title: Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae

doi: 10.1186/1475-2859-13-22

Figure Lengend Snippet: Yeast vector pFDC-BiP for the expression of human BiP. pFDC vector contains one inducible expression cassette under control of Saccharomyces cerevisiae GAL7 promoter with corresponding transcription terminator and the other cassette under control of constitutive S. cerevisiae promoter PGK1 with corresponding transcription terminator. 2 μm DNA, 1.74 kb fragment of yeast 2 μm DNA. PGK1-P, PGK1 gene promoter ( - 1 to - 541 bp); PGK1-T, PGK1 gene transcription terminator (371 bp). GAL7-P, GAL7 gene promoter ( - 1 to - 716 nt); GAL7-T, GAL7 gene transcription terminator (381 bp); FDH1, FDH1 gene of Candida maltosa , conferring resistance to formaldehyde; bla – beta lactamase gene, conferring resistance to ampicillin; BiP – human BiP protein coding gene ( HSPA5 , GenBank: AF216292).

Article Snippet: Recombinant human BiP protein purified from E. coli was purchased from StressMarq Biosciences Inc. (cat. no. SPR-107A).

Techniques: Plasmid Preparation, Expressing

Journal: Microbial Cell Factories

Article Title: Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae

doi: 10.1186/1475-2859-13-22

Figure Lengend Snippet: Secretion of human BiP protein into the yeast growth medium. SDS-PAGE (A) and Western blot against human BiP protein (B) of crude growth medium ( 40 × concentrated) of yeast cells carrying control vector pFDC or pFDC-BiP for expression of human BiP. Cells were incubated for 36 h in YEPD medium. M – prestained molecular ladder (ThermoScientific, cat. no. 26618).

Article Snippet: Recombinant human BiP protein purified from E. coli was purchased from StressMarq Biosciences Inc. (cat. no. SPR-107A).

Techniques: SDS Page, Western Blot, Plasmid Preparation, Expressing, Incubation

Journal: Microbial Cell Factories

Article Title: Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae

doi: 10.1186/1475-2859-13-22

Figure Lengend Snippet: Purification of secreted recombinant human BiP protein from yeast culture medium. M – unstained protein ladder (ThermoScientific, cat. no. 26614). A – crude yeast growth medium (20× concentrated), B – yeast growth medium after microfiltration (20× concentrated), C – 20× concentrated proteins from yeast growth medium in binding buffer after tangential ultrafiltration; D – purified yeast-derived recombinant human BiP protein (5 μg).

Article Snippet: Recombinant human BiP protein purified from E. coli was purchased from StressMarq Biosciences Inc. (cat. no. SPR-107A).

Techniques: Purification, Recombinant, Binding Assay, Derivative Assay

Journal: Microbial Cell Factories

Article Title: Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae

doi: 10.1186/1475-2859-13-22

Figure Lengend Snippet: Peptide mass fingerprinting of S. cerevisiae secreted GRP78/BiP protein by MALDI-TOF/TOF tandemic MS/MS together with UPLC/MS E method.

Article Snippet: Recombinant human BiP protein purified from E. coli was purchased from StressMarq Biosciences Inc. (cat. no. SPR-107A).

Techniques: Peptide Mass Fingerprinting, Tandem Mass Spectroscopy

Journal: Microbial Cell Factories

Article Title: Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae

doi: 10.1186/1475-2859-13-22

Figure Lengend Snippet: ESI-MS of recombinant human BiP purified from S. cerevisiae.

Article Snippet: Recombinant human BiP protein purified from E. coli was purchased from StressMarq Biosciences Inc. (cat. no. SPR-107A).

Techniques: Recombinant, Purification

Journal: Microbial Cell Factories

Article Title: Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae

doi: 10.1186/1475-2859-13-22

Figure Lengend Snippet: Native PAGE of recombinant human BiP. 5 μg of purified recombinant human BiP was loaded on gel. BSA was loaded as molecular weight marker.

Article Snippet: Recombinant human BiP protein purified from E. coli was purchased from StressMarq Biosciences Inc. (cat. no. SPR-107A).

Techniques: Clear Native PAGE, Recombinant, Purification, Molecular Weight, Marker

Journal: Microbial Cell Factories

Article Title: Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae

doi: 10.1186/1475-2859-13-22

Figure Lengend Snippet: Partial proteolysis of recombinant BiP with proteinase K in the presence of nucleotides. M – prestained protein ladder (ThermoScientific, cat. no. 26616). Undigested recombinant BiP (A), or digested with proteinase K without nucleotides (B), in the presence of 100 μM ATP (C) or 100 μM ADP (D).

Article Snippet: Recombinant human BiP protein purified from E. coli was purchased from StressMarq Biosciences Inc. (cat. no. SPR-107A).

Techniques: Recombinant

Journal: Microbial Cell Factories

Article Title: Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae

doi: 10.1186/1475-2859-13-22

Figure Lengend Snippet: ATPase activity of yeast-secreted human BiP. The amount of released phosphate by 1 μg of either bacterial or yeast-derived human BiP was determined after incubation at 25°C for 75 min. with a non-radioactive procedure. Values are the mean of three separate experiments with an error bar representing SD.

Article Snippet: Recombinant human BiP protein purified from E. coli was purchased from StressMarq Biosciences Inc. (cat. no. SPR-107A).

Techniques: Activity Assay, Derivative Assay, Incubation

Journal: Microbial Cell Factories

Article Title: Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae

doi: 10.1186/1475-2859-13-22

Figure Lengend Snippet: Evaluation of amount of intracellular human BiP protein in yeast S. cerevisiae cells. (A) SDS–PAGE of crude yeast lysates and indicated amounts of purified BiP; (B) Western blot using polyclonal antibodies against human BiP. M – prestained protein ladder (ThermoScientific, cat. no. 26618). pFDC and pFDC-hBiP – crude lysates (10 μg of whole cell protein in each lane) of yeast cells transformed with pFDC vector and pFDC-hBiP plasmid, respectively. 50, 75, 100, 150, 200, 300, 400 – amounts in nanograms of purified secreted human BiP protein loaded on gel.

Article Snippet: Recombinant human BiP protein purified from E. coli was purchased from StressMarq Biosciences Inc. (cat. no. SPR-107A).

Techniques: SDS Page, Purification, Western Blot, Transformation Assay, Plasmid Preparation