antibodies rabbit grp78  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: GRP78/BiP determines senescence evasion cell fate after cisplatin-based chemotherapy

doi: 10.1038/s41598-021-01540-8

Figure Lengend Snippet: CDDP-induced SASP in H460 and non-cytoprotective unfolded protein response. ( a ) Time-course analysis of the relative mRNA levels of various SASP genes by real-time Q-PCR in H460 cells treated with CDDP at 5 µM. ( b ) Protein expression levels of ER stress markers were detected in CDDP induced H460 senescence cells during SASP by western blot analysis (the full-length blots are presented in Supplementary Figures) ( c ) Time-course analysis of the relative mRNA levels of GRP78, CHOP and ATF-6 by real-time Q-PCR in H460 cells treated with CDDP at 5 µM. ( d ) H460 cells were treated with 5 µM CDDP at the respective days indicated. The cDNA from total RNA from these cells was subjected to PCR and Pst1 restriction digestion (CTGCA|G) to semi-quantitatively evaluate the levels of XBP1s . XBP1u unspliced XBP1 , XBP1s spliced XBP1 . For all the data, the average of three independent experiments is shown. Data was analyzed by two-tailed Student’s t-test. *Denotes p < 0.05.

Article Snippet: The transferred membrane was blocked with 5% nonfat milk in TBST (25 mM Tris–HCl (pH 7.5), 125 mM NaCl, 0.05% Tween 20) for about 1 h and incubated with primary antibodies rabbit GRP78 (78 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3177) and ER stress markers such as rabbit PERK (140 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:5683), rabbit IRE1α (130 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3294), rabbit pIRE1α (phosphor S724) (110 kDa, 1:1000 in 5% w/v BSA in TBST, abcam, ab48187), rabbit PDI (57 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3501), rabbit Ero1-Lα (60 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3264), mouse CHOP (27 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:2895), rabbit ATF-6 (D4Z8V) (100 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:65880) for overnight at 4 °C.

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: Scientific Reports

Article Title: GRP78/BiP determines senescence evasion cell fate after cisplatin-based chemotherapy

doi: 10.1038/s41598-021-01540-8

Figure Lengend Snippet: GRP78 primes the senescence cells for cell cycle re-entry when CDDP was removed. ( a ) Time-course analysis of the GRP78 protein expression in H460, A549 cells treated with CDDP at 5 µM. ( b ) Representative proliferation curves of H460, A549 cells treated with CDDP for 6 days then untreated for the remaining days (release) until day 18 ( c ) GRP78 Protein levels in H460 CRISPR-P131 cells by western blot analysis. ( d ) GRP78 Protein levels in H460 GRP78-OE cells selected by G418 (the full-length blots are presented in Supplementary Figures). ( e ) Proliferation assay of H460 CRISPR-P131, H460 GRP78-OE cells compare with control by monitoring for consecutive 6 days. (initial cell concentration is 10 3 cells/well) ( f,g ) Cell morphology and senescence-associated β-galactosidase staining of H460 CRISPR-P131 and H460 GRP78-OE cells. Data was presented as representative curves of at least three independent experiments. The scale bar in figure ( f,g ) is 100 µm. *Denotes p < 0.05, **Denotes p < 0.01.

Article Snippet: The transferred membrane was blocked with 5% nonfat milk in TBST (25 mM Tris–HCl (pH 7.5), 125 mM NaCl, 0.05% Tween 20) for about 1 h and incubated with primary antibodies rabbit GRP78 (78 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3177) and ER stress markers such as rabbit PERK (140 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:5683), rabbit IRE1α (130 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3294), rabbit pIRE1α (phosphor S724) (110 kDa, 1:1000 in 5% w/v BSA in TBST, abcam, ab48187), rabbit PDI (57 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3501), rabbit Ero1-Lα (60 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3264), mouse CHOP (27 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:2895), rabbit ATF-6 (D4Z8V) (100 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:65880) for overnight at 4 °C.

Techniques: Expressing, CRISPR, Western Blot, Proliferation Assay, Control, Concentration Assay, Staining

Journal: Scientific Reports

Article Title: GRP78/BiP determines senescence evasion cell fate after cisplatin-based chemotherapy

doi: 10.1038/s41598-021-01540-8

Figure Lengend Snippet: GRP78 is required for Bortezomib (BTZ) resistance senescence cells. ( a–c ) IC 50 value for H460 cells and CDDP6d cells with different concentration of ( a ) Tg (0.5–64 µM). ( b ) CDDP (2.5–160 µM). ( c ) Bortezomib (0.16–15.62 µM). ( d ) Bortezomib in A549. ( e ) Bortezomib in H460 GRP78-OE ( f ) Bortezomib in CRISPR-P131 ( g ) The level of GRP78 protein expression in A549 and H460 cells treated with BTZ at indicated concentrations. ( h ) IC 50 value for A549 treated with IT-139 (6.25–200 µM). ( i ) The level of GRP78 protein expression in A549-CDDP6d in the presence of IT-139 at indicated concentrations. ( j ) IT-139 treatment scheme for cell viability. ( k ) IC 50 value of Bortezomib treated in WT A549, A549-CDDP6d and A549-CDDP6d pretreated with 12.5 μM IT-139. ( l ) IT-139 treatment scheme for cell proliferation. ( m ) Cell proliferation of A549-CDDP6d with IT-139 treatment versus control. ( n ) GRP78 protein expression in A549-CDDP6d after Bortezomib treatment in the presence of IT-139 (the full-length blots are presented in Supplementary Figures). ( o ) IC 50 value of Bortezomib treated in H460-CDDP6d versus H460-GRP78-OE-CDDP6d. ( p ) Cell proliferation of H460 CDDP6d versus H460 GRP78-OE-CDDP6d after CDDP removal. ( q ) The level of CHOP protein expression in H460-CDDP6d and A549-CDDP6d in the presence of Bortezomib at indicated concentrations. ( r ) Comparison of CHOP protein expression from the band intensity relative to their respective control normalized with β-actin. For all the data, the average of three independent experiments is shown. Denotes p < 0.05.

Article Snippet: The transferred membrane was blocked with 5% nonfat milk in TBST (25 mM Tris–HCl (pH 7.5), 125 mM NaCl, 0.05% Tween 20) for about 1 h and incubated with primary antibodies rabbit GRP78 (78 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3177) and ER stress markers such as rabbit PERK (140 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:5683), rabbit IRE1α (130 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3294), rabbit pIRE1α (phosphor S724) (110 kDa, 1:1000 in 5% w/v BSA in TBST, abcam, ab48187), rabbit PDI (57 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3501), rabbit Ero1-Lα (60 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3264), mouse CHOP (27 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:2895), rabbit ATF-6 (D4Z8V) (100 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:65880) for overnight at 4 °C.

Techniques: Concentration Assay, CRISPR, Expressing, Control, Comparison

Journal: Scientific Reports

Article Title: GRP78/BiP determines senescence evasion cell fate after cisplatin-based chemotherapy

doi: 10.1038/s41598-021-01540-8

Figure Lengend Snippet:

Article Snippet: The transferred membrane was blocked with 5% nonfat milk in TBST (25 mM Tris–HCl (pH 7.5), 125 mM NaCl, 0.05% Tween 20) for about 1 h and incubated with primary antibodies rabbit GRP78 (78 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3177) and ER stress markers such as rabbit PERK (140 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:5683), rabbit IRE1α (130 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3294), rabbit pIRE1α (phosphor S724) (110 kDa, 1:1000 in 5% w/v BSA in TBST, abcam, ab48187), rabbit PDI (57 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3501), rabbit Ero1-Lα (60 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3264), mouse CHOP (27 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:2895), rabbit ATF-6 (D4Z8V) (100 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:65880) for overnight at 4 °C.

Techniques:

Journal: Scientific Reports

Article Title: GRP78/BiP determines senescence evasion cell fate after cisplatin-based chemotherapy

doi: 10.1038/s41598-021-01540-8

Figure Lengend Snippet:

Article Snippet: The transferred membrane was blocked with 5% nonfat milk in TBST (25 mM Tris–HCl (pH 7.5), 125 mM NaCl, 0.05% Tween 20) for about 1 h and incubated with primary antibodies rabbit GRP78 (78 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3177) and ER stress markers such as rabbit PERK (140 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:5683), rabbit IRE1α (130 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3294), rabbit pIRE1α (phosphor S724) (110 kDa, 1:1000 in 5% w/v BSA in TBST, abcam, ab48187), rabbit PDI (57 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3501), rabbit Ero1-Lα (60 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:3264), mouse CHOP (27 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:2895), rabbit ATF-6 (D4Z8V) (100 kDa, 1:1000 in 5% w/v BSA in TBST, cell signaling, cat no:65880) for overnight at 4 °C.

Techniques:

Journal: Scientific reports

Article Title: IL-1α is a DNA damage sensor linking genotoxic stress signaling to sterile inflammation and innate immunity.

doi: 10.1038/srep14756

Figure Lengend Snippet: Figure 1. IL-1α is recruited to DNA damage sites and secreted after genotoxic stress. (a) Human HT1080 fibrosarcoma and HaCaT human keratinocytes were subjected to genotoxic stresses: UV irradiation (5 mJ/cm2), 10 mM H2O2 or 50 μ gr/ml Bleomycin. 16 h post exposure, hIL-1α ELISA was used to measure secreted IL-1α in cell supernatants. All experiments were performed in triplicates and data are expressed as mean ± SD. (b) Nuclear/cytoplasmic re-localization of IL-1α after DNA damage. Live cell imaging of B16 melanoma cells expressing GFP-IL-1α during treatment with 100 μM H2O2. Images were collected every 30 min for a period of 24 h. Representative images from indicated time points are shown (for full video see Supplementary Video 1, for averaged fluorescence intensities see also Supplementary Figure 1b) White scale bars, 20 μm. (c,d) Nuclear IL-1α co-localizes with γ H2AX foci after genotoxic stress. (c) B16 melanoma cells expressing GFP-IL-1α were treated with Etoposide 10 µg/mL for 2 h or (d) microirradiated with femtosecond laser pulses at λ = 775 nm (see also Supplementary Videos 2 and 3). After fixation of cells, detection of GFP-IL-1α , DAPI or immunostaining of γ H2AX was preformed and visualized by confocal microscopy. White scale bars, 20 μm (e) Recruitment kinetics of IL-1α to DNA damage sites. B16 melanoma cells expressing IL-1α –GFP were laser-microirradiated along a single line to induce DNA damage. Fluorescence intensity in the damaged region was measured up to 15 min from irradiaton in 1 min intervals. Data is expressed as mean ± SEM of increase in fluorescence intensity (n = 10 cells). (f) IL-1α localizes to Cyclobutane Pyrimidine Dimers (CPD) induced via laser microirradiation. B16 melanoma cells expressing GFP-IL-1α were laser irradiated and CPDs were visualized by immunostaining using specific antibodies. White scale bars, 20 μm.

Article Snippet: The cells were allowed to express GFP-IL-1α and left to recover for an additional 24 h. To induce DNA damage, UV was applied as described above and levels of secreted GFP-IL-1α were measured by measuring GFP using the PathScan total GFP Sandwich ELISA kit (Cell signaling).

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay, Live Cell Imaging, Expressing, Fluorescence, Immunostaining, Confocal Microscopy

Journal: Scientific reports

Article Title: IL-1α is a DNA damage sensor linking genotoxic stress signaling to sterile inflammation and innate immunity.

doi: 10.1038/srep14756

Figure Lengend Snippet: Figure 3. IL-1α acetylation within the nuclear localization sequence impacts on IL1α subcellular localisation. (a) IL-1α precursor is recognized by a pan acetyl antibody. Endogenous IL-1α was immunoprecipitated (IP) from nuclear extracts of Raw 264.7 cells, either induced or non-induced with 100 ng/ml LPS. Total IP proteins were separated over 15% SDS PAGE, transferred to nitrocellulose membranes and blotted with anti-mouse IL-1α (top panel) or anti-Kac (bottom panel). Acetylated IL- 1α is marked by arrows and IP antibody light and heavy chain signals are indicated. (b) Annotated MS/ MS spectrum of the tryptic peptide VTVSATSSN(Deam)GK(Acetyl)ILK (MH2 + 724.40 Da) showing acetylation of IL-1α (Uniprot ID P01582) at K82 and N80 deamidation. (c) PrecIL-1α K82 mutants affect IL-1α sub-cellular localization. Confocal microscopic analysis of GFP tagged WT IL-1α and mutations of precIL-1α K82 to glutamine (precIL-1α K82Q, mimicking acetylation) and to arginine (precIL-1α K82R non-acetylateable). White scale bars, 20 μm (d) IL-1α K82 mutations reduce cytokine secretion after DNA damage. Mouse B16 cells were transfected with the indicated GFP IL-1α plasmids. The cells were then subjected to 100 μ M H2O2. 16h after stress induction levels of secreted GFP IL-1α in cell growth medium was measured using a GFP ELISA. GFP IL-1α levels in cell lysates were used to normalize for transfection efficiencies and non-transfected cells were used as negative controls. Data are expressed as mean ± SD of three independent experiments. (e) Histone deacetylase inhibition by TSA increases IL-1α nuclear localization. Images of cells expressing GFP IL-1α either non-treated (control) or treated with TSA (100 ng/ml) were collected every hour for 22 h and representative images for three time points (0, 11 and 22 hours) are shown (For averaged fluorescence intensities of nuclear/cytoplasmic ratios see Supplementary Figure 1b). (f) HDAC-1 and IL-1α can co-localize at DNA damage lesions. Cells expressing GFP IL-1α were laser- microirradiated for the induction of DNA damage. Localization of HDAC-1 and IL-1α –GFP were visualized by confocal microscopic analysis.

Article Snippet: The cells were allowed to express GFP-IL-1α and left to recover for an additional 24 h. To induce DNA damage, UV was applied as described above and levels of secreted GFP-IL-1α were measured by measuring GFP using the PathScan total GFP Sandwich ELISA kit (Cell signaling).

Techniques: Sequencing, Immunoprecipitation, SDS Page, Tandem Mass Spectroscopy, Transfection, Enzyme-linked Immunosorbent Assay, Histone Deacetylase Assay, Inhibition, Expressing, Control, Fluorescence