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Boster Bio recombinant grp78 protein

CDDO‐2P‐Im binds <t>GRP78</t> 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.

Recombinant Grp78 Protein, supplied by Boster Bio, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The synthetic oleanane triterpenoid CDDO‐2P‐Im binds GRP78 / BiP to induce unfolded protein response‐mediated apoptosis in myeloma"

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

Journal: Molecular Oncology

doi: 10.1002/1878-0261.13447

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

Image Search Results

Experimental design. The timing and sequence of LC and GRP78 injections and procedures are shown. Red arrows, LC—microinjections of the proteasome inhibitor lactacystin into the SNpc (0.4 μg/1 μL). Black arrows, GRP78—treatment with recombinant human heat shock protein GRP78 (1.6 μg/8 μL) or the corresponding vehicle, sterile PBS, administered intranasally 4 h and 28 h following each microinjection of LC or vehicle and 7 days after the last injection.

Journal: International Journal of Molecular Sciences

Article Title: Intranasal Administration of GRP78 Protein (HSPA5) Confers Neuroprotection in a Lactacystin-Induced Rat Model of Parkinson’s Disease

doi: 10.3390/ijms25073951

Figure Lengend Snippet: Experimental design. The timing and sequence of LC and GRP78 injections and procedures are shown. Red arrows, LC—microinjections of the proteasome inhibitor lactacystin into the SNpc (0.4 μg/1 μL). Black arrows, GRP78—treatment with recombinant human heat shock protein GRP78 (1.6 μg/8 μL) or the corresponding vehicle, sterile PBS, administered intranasally 4 h and 28 h following each microinjection of LC or vehicle and 7 days after the last injection.

Article Snippet: Recombinant human heat shock protein GRP78 (Sigma, Livonia, MI, USA) was diluted in sterile PBS (pH 7.4) and administered intranasally (to each nostril) to rats ( n = 6–8, group LC+GRP78) at a dose of 1.6 μg/8 μL, 4 h and 28 h after each microinjection of LC.

Techniques: Sequencing, Recombinant, Sterility, Injection

Labeled GRP78 penetrates the brain and is localized in DA neurons and microglial cells of the substantia nigra pars compacta (SNpc) 3 h after its intranasal administration in a rat model of Parkinson’s disease. GRP78 protein labeled by Alexa-555 was administered intranasally to rats ( n = 4) after a microinjection of lactacystin as described in the Materials and Methods. Brain sections were stained with ( a ) specific anti-TH antibodies (green signal), ( b ) anti-Grp78 antibodies specific to human protein (green signal), and ( c ) anti-Iba-1 antibodies (green signal). ( d – f ) Localization of labeled GRP78 is seen as a red signal. ( g – i ) Panels show co-localization of labeled GRP78 and anti-TH, anti-Grp78, or anti-Iba-1 signals. ( j – l ) Panels show magnified representative images of the co-localization within neurons and microglia cells marked by yellow box. Arrows indicate co-localization of labeled GRP78 with corresponding proteins. Images were obtained using confocal microscopy. Scale bars are 25 μm for neurons in the SNpc and 10 μm for microglia.

Journal: International Journal of Molecular Sciences

Article Title: Intranasal Administration of GRP78 Protein (HSPA5) Confers Neuroprotection in a Lactacystin-Induced Rat Model of Parkinson’s Disease

doi: 10.3390/ijms25073951

Figure Lengend Snippet: Labeled GRP78 penetrates the brain and is localized in DA neurons and microglial cells of the substantia nigra pars compacta (SNpc) 3 h after its intranasal administration in a rat model of Parkinson’s disease. GRP78 protein labeled by Alexa-555 was administered intranasally to rats ( n = 4) after a microinjection of lactacystin as described in the Materials and Methods. Brain sections were stained with ( a ) specific anti-TH antibodies (green signal), ( b ) anti-Grp78 antibodies specific to human protein (green signal), and ( c ) anti-Iba-1 antibodies (green signal). ( d – f ) Localization of labeled GRP78 is seen as a red signal. ( g – i ) Panels show co-localization of labeled GRP78 and anti-TH, anti-Grp78, or anti-Iba-1 signals. ( j – l ) Panels show magnified representative images of the co-localization within neurons and microglia cells marked by yellow box. Arrows indicate co-localization of labeled GRP78 with corresponding proteins. Images were obtained using confocal microscopy. Scale bars are 25 μm for neurons in the SNpc and 10 μm for microglia.

Techniques: Labeling, Staining, Confocal Microscopy

Exogenous GRP78 prevents abnormal accumulation of α-syn phosphorylated at S129 (pS129) in nigral tissue in rat model of Parkinson’s disease. Nigral content of ( a ) soluble form of α-syn, ( b ) α-syn phosphorylated at S129 (pS129), ( c ) phosphorylated to soluble α-syn ratio. Western blot analysis of nigral tissue was conducted with the antibodies against soluble and S129-phosphorylated forms of α-syn. Anti-GAPDH antibody staining was used as the loading control. ( d ) Representative Western blots are shown in panel. The results are presented as percentages of the control (100%) in ( a – c ) panels. Bar charts indicate mean values with standard errors. The dots, squares, triangles and rhombus show individual values per rat. Two-way ANOVA test followed by Tukey’s post hoc analysis were performed to determine the effects of GRP78 therapy. Asterisks indicate significant differences between groups according to Tukey’s post hoc tests: * p < 0.05, *** p < 0.001, vs. the vehicle group; # p < 0.05, ## p < 0.01, vs. the LC group. Interaction factor for soluble α-syn F (1, 15) = 7.329 p = 0.0162; Grp78 factor F (1, 15) = 14.34 p = 0.0018; LC (lactacystin) factor F (1, 15) = 13.63 p = 0.0022; Interaction factor for pS129-α-syn F (1, 17) = 4.982 p = 0.0394; Grp78 factor F (1, 17) = 6.399 p = 0.0216; LC factor F (1, 17) = 14.16 p = 0.0016.

Journal: International Journal of Molecular Sciences

Article Title: Intranasal Administration of GRP78 Protein (HSPA5) Confers Neuroprotection in a Lactacystin-Induced Rat Model of Parkinson’s Disease

doi: 10.3390/ijms25073951

Figure Lengend Snippet: Exogenous GRP78 prevents abnormal accumulation of α-syn phosphorylated at S129 (pS129) in nigral tissue in rat model of Parkinson’s disease. Nigral content of ( a ) soluble form of α-syn, ( b ) α-syn phosphorylated at S129 (pS129), ( c ) phosphorylated to soluble α-syn ratio. Western blot analysis of nigral tissue was conducted with the antibodies against soluble and S129-phosphorylated forms of α-syn. Anti-GAPDH antibody staining was used as the loading control. ( d ) Representative Western blots are shown in panel. The results are presented as percentages of the control (100%) in ( a – c ) panels. Bar charts indicate mean values with standard errors. The dots, squares, triangles and rhombus show individual values per rat. Two-way ANOVA test followed by Tukey’s post hoc analysis were performed to determine the effects of GRP78 therapy. Asterisks indicate significant differences between groups according to Tukey’s post hoc tests: * p < 0.05, *** p < 0.001, vs. the vehicle group; # p < 0.05, ## p < 0.01, vs. the LC group. Interaction factor for soluble α-syn F (1, 15) = 7.329 p = 0.0162; Grp78 factor F (1, 15) = 14.34 p = 0.0018; LC (lactacystin) factor F (1, 15) = 13.63 p = 0.0022; Interaction factor for pS129-α-syn F (1, 17) = 4.982 p = 0.0394; Grp78 factor F (1, 17) = 6.399 p = 0.0216; LC factor F (1, 17) = 14.16 p = 0.0016.

Techniques: Western Blot, Staining

Exogenous GRP78 blocks the pro-apoptotic GRP78/eIF2α/CHOP/caspase-3,9 signaling pathway of the UPR in nigral tissue in a rat model of Parkinson’s disease. Nigral content of ( a ) GRP78, ( b ) phosphorylated to total eIF2α, ( c ) CHOP. ( d ) Representative immunoblots. Nigral content of ( e ) cleaved caspase-9, ( f ) cleaved caspase-3. ( g ) Representative immunoblots. Western blot analysis of the nigral tissue was conducted with the antibodies against GRP78 (1:1000, rabbit, Abcam, Cambridge, UK), eIF2a (1:750, rabbit, Affinity Biosciences, Zhenjiang, China), pSer51-eIF2a (1:1000, rabbit, Abcam, Cambridge, UK), CHOP (1:1000, rabbit, Affinity Biosciences, Zhenjiang, China), cleaved caspase-9 (1:1000, rabbit, Affinity Biosciences, Zhenjiang, China), and cleaved caspase-3 (1:1000, rabbit, Affinity Biosciences, Zhenjiang, China). Staining with anti-β-Actin antibodies (1:1000, mouse, Santa Cruz Biotechnology, Dallas, TX, USA) was used as the loading control. The results are presented as percentages of the control (panels ( a – c , e , f )). Bar charts indicate mean values with standard errors. The dots, squares, triangles and rhombus indicate individual values per rat. Two-way ANOVA test followed by Tukey’s post hoc analysis were performed to determine the effects of GRP78 therapy. Asterisks indicate significant differences between groups according to Tukey’s post hoc tests: * p < 0.05; ** p < 0.01. *** p < 0.001, vs. the vehicle group; # p < 0.05, ## p < 0.01, vs. the LC group. Interaction factor for GRP78 F (1, 20) = 8.83 p = 0.0076; Grp78 treatment factor F (1, 20) = 7.413 p = 0.0131; LC (lactacystin) factor F (1, 20) = 15.89 p = 0.0007. Interaction factor for p-eIF2a/eIF2a ratio F (1, 19) = 5.68 p = 0.0278; Grp78 treatment factor F (1, 19) = 1.874 p = 0.1869; LC factor F (1, 19) = 7.819 p = 0.0115. Interaction factor for CHOP F (1, 16) = 3.126 p = 0.0961; Grp78 treatment factor F (1, 16) = 3.672 p = 0.0734; LC factor F (1, 16) = 47.898 p = 0.0418. Interaction factor for cleaved caspase-9 F (1, 17) = 4.124 p = 0.0582; Grp78 treatment factor F (1, 17) = 1.513 p = 0.2355; LC factor F (1, 17) = 5.911 p = 0.0264. Interaction factor for cleaved caspase-3 F (1, 17) = 6.417 p = 0.0214; Grp78 treatment factor F (1, 17) = 5.646 p = 0.0295; LC factor F (1, 17) = 7.451 p = 0.0143.

Journal: International Journal of Molecular Sciences

Article Title: Intranasal Administration of GRP78 Protein (HSPA5) Confers Neuroprotection in a Lactacystin-Induced Rat Model of Parkinson’s Disease

doi: 10.3390/ijms25073951

Figure Lengend Snippet: Exogenous GRP78 blocks the pro-apoptotic GRP78/eIF2α/CHOP/caspase-3,9 signaling pathway of the UPR in nigral tissue in a rat model of Parkinson’s disease. Nigral content of ( a ) GRP78, ( b ) phosphorylated to total eIF2α, ( c ) CHOP. ( d ) Representative immunoblots. Nigral content of ( e ) cleaved caspase-9, ( f ) cleaved caspase-3. ( g ) Representative immunoblots. Western blot analysis of the nigral tissue was conducted with the antibodies against GRP78 (1:1000, rabbit, Abcam, Cambridge, UK), eIF2a (1:750, rabbit, Affinity Biosciences, Zhenjiang, China), pSer51-eIF2a (1:1000, rabbit, Abcam, Cambridge, UK), CHOP (1:1000, rabbit, Affinity Biosciences, Zhenjiang, China), cleaved caspase-9 (1:1000, rabbit, Affinity Biosciences, Zhenjiang, China), and cleaved caspase-3 (1:1000, rabbit, Affinity Biosciences, Zhenjiang, China). Staining with anti-β-Actin antibodies (1:1000, mouse, Santa Cruz Biotechnology, Dallas, TX, USA) was used as the loading control. The results are presented as percentages of the control (panels ( a – c , e , f )). Bar charts indicate mean values with standard errors. The dots, squares, triangles and rhombus indicate individual values per rat. Two-way ANOVA test followed by Tukey’s post hoc analysis were performed to determine the effects of GRP78 therapy. Asterisks indicate significant differences between groups according to Tukey’s post hoc tests: * p < 0.05; ** p < 0.01. *** p < 0.001, vs. the vehicle group; # p < 0.05, ## p < 0.01, vs. the LC group. Interaction factor for GRP78 F (1, 20) = 8.83 p = 0.0076; Grp78 treatment factor F (1, 20) = 7.413 p = 0.0131; LC (lactacystin) factor F (1, 20) = 15.89 p = 0.0007. Interaction factor for p-eIF2a/eIF2a ratio F (1, 19) = 5.68 p = 0.0278; Grp78 treatment factor F (1, 19) = 1.874 p = 0.1869; LC factor F (1, 19) = 7.819 p = 0.0115. Interaction factor for CHOP F (1, 16) = 3.126 p = 0.0961; Grp78 treatment factor F (1, 16) = 3.672 p = 0.0734; LC factor F (1, 16) = 47.898 p = 0.0418. Interaction factor for cleaved caspase-9 F (1, 17) = 4.124 p = 0.0582; Grp78 treatment factor F (1, 17) = 1.513 p = 0.2355; LC factor F (1, 17) = 5.911 p = 0.0264. Interaction factor for cleaved caspase-3 F (1, 17) = 6.417 p = 0.0214; Grp78 treatment factor F (1, 17) = 5.646 p = 0.0295; LC factor F (1, 17) = 7.451 p = 0.0143.

Techniques: Western Blot, Staining

Exogenous GRP78 inhibits microglia activation in a lactacystin rat model of Parkinson’s disease. ( a ) Brain sections (10 µm) of the substantia nigra pars compacta (SNpc, ( a )) were prepared according to the brain atlas and stained with antibodies against Iba-1 (1:500; rabbit, Novus Biologicals, Centennial, CO, USA). The images were obtained using a Zeiss Axio Imager A1 microscope (Carl Zeiss, Oberkochen, Germany) with a built-in video camera and Axio-Vision 4.8 software. Original images are shown in the upper panel. Scale bars are 100 μm. The second panel show magnified images of microglia cells (zoom). The third panel show magnified images of microglia morphology of cells within dotted box area (zoom). ( b ) Quantitative analysis was performed using 10–12 sections from each animal at the same level of the studied zones, separated by approximately 70 μm. The number of cells accounted for a standard area of tissue captured by a light microscope camera using ×20 lens. The analysis was performed using the PhotoM freeware version 1.21 ( http://www.t_lambda.chat.ru/ accessed on 11 December 2019). Bar charts indicate mean values with standard errors. The dots, squares, triangles and rhombus show individual values per rat. Two-way ANOVA test followed by Tukey’s post hoc analysis were performed to determine the effects of GRP78 therapy. Asterisks indicate significant differences between groups according to Tukey’s post hoc tests: ** p < 0.01 vs. the vehicle group; # p < 0.05 vs. the LC group. Interaction factor for microglia in SNpc F (2, 23) = 2.099; Grp78 factor F (2, 23) = 5.466 p = 0.0284; LC factor F (2, 23) = 17.04 p = 0.0004.

Journal: International Journal of Molecular Sciences

Article Title: Intranasal Administration of GRP78 Protein (HSPA5) Confers Neuroprotection in a Lactacystin-Induced Rat Model of Parkinson’s Disease

doi: 10.3390/ijms25073951

Figure Lengend Snippet: Exogenous GRP78 inhibits microglia activation in a lactacystin rat model of Parkinson’s disease. ( a ) Brain sections (10 µm) of the substantia nigra pars compacta (SNpc, ( a )) were prepared according to the brain atlas and stained with antibodies against Iba-1 (1:500; rabbit, Novus Biologicals, Centennial, CO, USA). The images were obtained using a Zeiss Axio Imager A1 microscope (Carl Zeiss, Oberkochen, Germany) with a built-in video camera and Axio-Vision 4.8 software. Original images are shown in the upper panel. Scale bars are 100 μm. The second panel show magnified images of microglia cells (zoom). The third panel show magnified images of microglia morphology of cells within dotted box area (zoom). ( b ) Quantitative analysis was performed using 10–12 sections from each animal at the same level of the studied zones, separated by approximately 70 μm. The number of cells accounted for a standard area of tissue captured by a light microscope camera using ×20 lens. The analysis was performed using the PhotoM freeware version 1.21 ( http://www.t_lambda.chat.ru/ accessed on 11 December 2019). Bar charts indicate mean values with standard errors. The dots, squares, triangles and rhombus show individual values per rat. Two-way ANOVA test followed by Tukey’s post hoc analysis were performed to determine the effects of GRP78 therapy. Asterisks indicate significant differences between groups according to Tukey’s post hoc tests: ** p < 0.01 vs. the vehicle group; # p < 0.05 vs. the LC group. Interaction factor for microglia in SNpc F (2, 23) = 2.099; Grp78 factor F (2, 23) = 5.466 p = 0.0284; LC factor F (2, 23) = 17.04 p = 0.0004.

Techniques: Activation Assay, Staining, Microscopy, Software, Light Microscopy

Exogenous GRP78 inhibits the production of pro-inflammatory cytokines TNF-α and IL-6 in a lactacystin rat model of Parkinson’s disease. Content of ( a ) TNF-α and ( b ) IL-6 in SNpc tissue in the LC-induced model of PD. ( c , d ) Representative Western blots. Western blot analysis was conducted with the antibodies against TNF-α (1:1000, rabbit, Affinity Biosciences, Zhenjiang, China), IL-6 (1:1000, rabbit, Affinity Biosciences, Zhenjiang, China). Anti-GAPDH antibodies (1:1000, mouse, Affinity Biosciences, Zhenjiang, China) were used for loading control. Bar charts indicate mean values with standard errors. The dots, squares, triangles and rhombus show individual values per rat. Two-way ANOVA test followed by Tukey’s post hoc analysis were performed to determine the effects of GRP78 therapy. Asterisks indicate significant differences between groups according to Tukey’s post hoc tests: * p < 0.05, *** p < 0.001, vs. the vehicle group; # p < 0.05, vs. the LC group. Interaction factor for TNF-α F (1, 17) = 3.228 p = 0.0902; Grp78 treatment factor F (1, 17) = 2.034 p = 0.1719; LC (lactacystin) factor F (1, 17) = 7.614 p = 0.0134. Interaction factor for IL-6 F (1, 17) = 4.896 p = 0.0409; Grp78 treatment factor F (1, 17) = 3.164 p = 0.0932; F (1, 17) = 19.58 p = 0.0004.

Journal: International Journal of Molecular Sciences

Article Title: Intranasal Administration of GRP78 Protein (HSPA5) Confers Neuroprotection in a Lactacystin-Induced Rat Model of Parkinson’s Disease

doi: 10.3390/ijms25073951

Figure Lengend Snippet: Exogenous GRP78 inhibits the production of pro-inflammatory cytokines TNF-α and IL-6 in a lactacystin rat model of Parkinson’s disease. Content of ( a ) TNF-α and ( b ) IL-6 in SNpc tissue in the LC-induced model of PD. ( c , d ) Representative Western blots. Western blot analysis was conducted with the antibodies against TNF-α (1:1000, rabbit, Affinity Biosciences, Zhenjiang, China), IL-6 (1:1000, rabbit, Affinity Biosciences, Zhenjiang, China). Anti-GAPDH antibodies (1:1000, mouse, Affinity Biosciences, Zhenjiang, China) were used for loading control. Bar charts indicate mean values with standard errors. The dots, squares, triangles and rhombus show individual values per rat. Two-way ANOVA test followed by Tukey’s post hoc analysis were performed to determine the effects of GRP78 therapy. Asterisks indicate significant differences between groups according to Tukey’s post hoc tests: * p < 0.05, *** p < 0.001, vs. the vehicle group; # p < 0.05, vs. the LC group. Interaction factor for TNF-α F (1, 17) = 3.228 p = 0.0902; Grp78 treatment factor F (1, 17) = 2.034 p = 0.1719; LC (lactacystin) factor F (1, 17) = 7.614 p = 0.0134. Interaction factor for IL-6 F (1, 17) = 4.896 p = 0.0409; Grp78 treatment factor F (1, 17) = 3.164 p = 0.0932; F (1, 17) = 19.58 p = 0.0004.

Techniques: Western Blot

GRP78 can protect neurons from the excessive activation of microglia via NF-κB signaling pathways in the LC-induced rat model of PD. Content of ( a ) NF-κB p65, ( b ) phosphorylated NF-κB p65 (Ser536), and ( c ) phosphorylated to total NF-κB p65 ratio in SNpc in LC model of PD. ( d ) Representative immunoblots. Western blot analysis of the nigral tissue was conducted with the antibodies against NF-κB p65 (1:1000, mouse, Cell Signaling, Danvers, MA, USA), and phosphorylated NF-κB p65 (Ser536) (1:1000, rabbit, Cell Signaling, Danvers, MA, USA). Anti-GAPDH antibodies (1:1000, mouse, Affinity Biosciences, China) were used for loading control. Bar charts indicate mean values with standard errors. The dots, squares, triangles and rhombus show individual values per rat. Two-way ANOVA test followed by Tukey’s post hoc analysis were performed to determine the effects of GRP78 therapy. Asterisks indicate significant differences between groups according to Tukey’s post hoc tests: * p < 0.05, ** p < 0.01, vs. the vehicle group; # p < 0.05 vs. the LC group. Interaction factor for total NF-κB F (1, 17) = 2.337 p = 0.1447; Grp78 treatment factor F (1, 17) = 0.3563 p = 0.5584; LC factor F (1, 17) = 3.978 p = 0.0624. Interaction factor for phosphorylated NF-κB (Ser536) F (1, 17) = 3.776 p = 0.0687; Grp78 treatment factor F (1, 17) = 3.897 p = 0.0648; LC factor F (1, 17) = 10.29 p = 0.0052. Interaction factor for p-NF-κB (Ser536)/NF-κB ratio F (1, 17) = 5.358 p = 0.0334; Grp78 treatment factor F (1, 17) = 1.339 p = 0.2631; LC factor F (1, 17) = 2.477 p = 0.1339.

Journal: International Journal of Molecular Sciences

Article Title: Intranasal Administration of GRP78 Protein (HSPA5) Confers Neuroprotection in a Lactacystin-Induced Rat Model of Parkinson’s Disease

doi: 10.3390/ijms25073951

Figure Lengend Snippet: GRP78 can protect neurons from the excessive activation of microglia via NF-κB signaling pathways in the LC-induced rat model of PD. Content of ( a ) NF-κB p65, ( b ) phosphorylated NF-κB p65 (Ser536), and ( c ) phosphorylated to total NF-κB p65 ratio in SNpc in LC model of PD. ( d ) Representative immunoblots. Western blot analysis of the nigral tissue was conducted with the antibodies against NF-κB p65 (1:1000, mouse, Cell Signaling, Danvers, MA, USA), and phosphorylated NF-κB p65 (Ser536) (1:1000, rabbit, Cell Signaling, Danvers, MA, USA). Anti-GAPDH antibodies (1:1000, mouse, Affinity Biosciences, China) were used for loading control. Bar charts indicate mean values with standard errors. The dots, squares, triangles and rhombus show individual values per rat. Two-way ANOVA test followed by Tukey’s post hoc analysis were performed to determine the effects of GRP78 therapy. Asterisks indicate significant differences between groups according to Tukey’s post hoc tests: * p < 0.05, ** p < 0.01, vs. the vehicle group; # p < 0.05 vs. the LC group. Interaction factor for total NF-κB F (1, 17) = 2.337 p = 0.1447; Grp78 treatment factor F (1, 17) = 0.3563 p = 0.5584; LC factor F (1, 17) = 3.978 p = 0.0624. Interaction factor for phosphorylated NF-κB (Ser536) F (1, 17) = 3.776 p = 0.0687; Grp78 treatment factor F (1, 17) = 3.897 p = 0.0648; LC factor F (1, 17) = 10.29 p = 0.0052. Interaction factor for p-NF-κB (Ser536)/NF-κB ratio F (1, 17) = 5.358 p = 0.0334; Grp78 treatment factor F (1, 17) = 1.339 p = 0.2631; LC factor F (1, 17) = 2.477 p = 0.1339.

Techniques: Activation Assay, Western Blot

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.

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

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