rabbit anti human bip grp78  (Proteintech)

Journal: Liver Research

Article Title: Combination of brefeldin A and tunicamycin induces apoptosis in HepG2 cells through the endoplasmic reticulum stress-activated PERK-eIF2α-ATF4-CHOP signaling pathway

doi: 10.1016/j.livres.2025.01.004

Figure Lengend Snippet: Antibodies used for Western blots.

Article Snippet: Rabbit anti-human BiP/GRP78 , Protein Tech, China , 66574-1-Ig , 1:1000.

Techniques: Western Blot

Journal: International Journal of Oncology

Article Title: Galectin-1 binds GRP78 to promote the proliferation and metastasis of gastric cancer

doi: 10.3892/ijo.2022.5431

Figure Lengend Snippet: Associations between Gal-1 expression and clinicopathological features of 80 patients with gastric cancer.

Article Snippet: The cells were permeabilized with 0.1% Triton X-100 for 15 min. After blocking with 5% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature for 60 min, the cells were incubated with the following antibodies for 60 min at 37°C: Rabbit anti-human GRP78 (1:300; cat. no. 11587-1-AP) and mouse anti-human Gal-1 (1:100; cat. no. 60223-1-Ig; both from ProteinTech Group, Inc.).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: Galectin-1 binds GRP78 to promote the proliferation and metastasis of gastric cancer

doi: 10.3892/ijo.2022.5431

Figure Lengend Snippet: Expression of GRP78 in gastric cancer with different clinicopathological variables using our own cohort.

Article Snippet: The cells were permeabilized with 0.1% Triton X-100 for 15 min. After blocking with 5% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature for 60 min, the cells were incubated with the following antibodies for 60 min at 37°C: Rabbit anti-human GRP78 (1:300; cat. no. 11587-1-AP) and mouse anti-human Gal-1 (1:100; cat. no. 60223-1-Ig; both from ProteinTech Group, Inc.).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: Galectin-1 binds GRP78 to promote the proliferation and metastasis of gastric cancer

doi: 10.3892/ijo.2022.5431

Figure Lengend Snippet: GRP78 is associated with Gal-1. (A) The interaction protein network of Gal-1 was revealed by the BioGRID database ( https://thebiogrid.org/ ). (B) GRP78 mRNA expression in GC (n=408) and average (n=211) samples were evaluated using the online tool GEPIA. (C) The expression level of Gal-1 mRNA is independent of GRP78 mRNA in The Cancer Genome Atlas database. (D and E) Gal-1 physically interacted with GRP78. The endogenous proteins from HGC-27 cells were immunoprecipitated with IgG or antibodies against Gal-1 and GRP78, followed by western blot analysis and cell lysis for input. (F) Immunofluorescence colocalization of Gal-1 and GRP78 in GC cells (scale bar, 10 µ m). (G) A western blot assay was performed to detect the GRP78 protein levels in the HGC-27 cells, knocking-down Gal-1. (H) Western blot analysis was performed to detect the GRP78 protein levels in the AGS cells overexpressing Gal-1. * P<0.05. GRP78, glucose-regulated protein 78; Gal-1, galectin-1; GC, gastric cancer; sh-, short hairpin; OE, overexpression.

Article Snippet: The cells were permeabilized with 0.1% Triton X-100 for 15 min. After blocking with 5% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature for 60 min, the cells were incubated with the following antibodies for 60 min at 37°C: Rabbit anti-human GRP78 (1:300; cat. no. 11587-1-AP) and mouse anti-human Gal-1 (1:100; cat. no. 60223-1-Ig; both from ProteinTech Group, Inc.).

Techniques: Expressing, Immunoprecipitation, Western Blot, Lysis, Immunofluorescence, Over Expression

Journal: International Journal of Oncology

Article Title: Galectin-1 binds GRP78 to promote the proliferation and metastasis of gastric cancer

doi: 10.3892/ijo.2022.5431

Figure Lengend Snippet: (A) The IHC assay of Gal-1 and GRP78 expression in GC tissues and corresponding adjacent normal tissues was performed, and the representative images were shown. (B) Qualification of GRP78 staining in TMAs described in panel A. The graph depicts the total score, the multiplication product of staining intensity and the percentage of stained cells. (C) Statistical analysis of the relationship between the expression level of GRP78 and Gal-1 (P-value: Spearman's correlation coefficient). (D) Kaplan-Meier overall survival curves for all 80 patients with GC stratified by high and low expression of GRP78. ** P<0.01. Gal-1, galectin-1; GRP78, glucose-regulated protein 78; IHC, immunohistochemical; GC, gastric cancer.

Article Snippet: The cells were permeabilized with 0.1% Triton X-100 for 15 min. After blocking with 5% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature for 60 min, the cells were incubated with the following antibodies for 60 min at 37°C: Rabbit anti-human GRP78 (1:300; cat. no. 11587-1-AP) and mouse anti-human Gal-1 (1:100; cat. no. 60223-1-Ig; both from ProteinTech Group, Inc.).

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: International Journal of Oncology

Article Title: Galectin-1 binds GRP78 to promote the proliferation and metastasis of gastric cancer

doi: 10.3892/ijo.2022.5431

Figure Lengend Snippet: Tumor-promoting effect of Gal-1 is dependent on the upregulation of GRP78. (A) The knockdown and overexpression efficiency of GRP78 in AGS and HGC-27 cells was confirmed by western blot analysis. (B) Colony formation assay revealed that ectopic GRP78 expression restored the number of cell colonies in shGal-1HGC-27 cells. (C) Ectopic GRP78 expression promoted migration and invasion in shGal-1 HGC-27 cells. (D) Colony formation assay showed that lentivirus-mediated knockdown of GRP78, and ectopic Gal-1 expression inhibited cell growth of AGS cells. (E) Knockout GRP78 inhibited migration and invasion in OE-Gal-1 AGS cells. * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001. Gal-1, galectin-1; GRP78, glucose-regulated protein 78; sh-, short hairpin; OE, overexpression.

Article Snippet: The cells were permeabilized with 0.1% Triton X-100 for 15 min. After blocking with 5% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature for 60 min, the cells were incubated with the following antibodies for 60 min at 37°C: Rabbit anti-human GRP78 (1:300; cat. no. 11587-1-AP) and mouse anti-human Gal-1 (1:100; cat. no. 60223-1-Ig; both from ProteinTech Group, Inc.).

Techniques: Knockdown, Over Expression, Western Blot, Colony Assay, Expressing, Migration, Knock-Out

Journal: Science Advances

Article Title: RIPK3-mediated inflammation is a conserved β cell response to ER stress

doi: 10.1126/sciadv.abd7272

Figure Lengend Snippet: ( A ) β cell–protective effect of a RIPK1 inhibitor (Nec-1) and a RIPK3 inhibitor (GSK’872). Data represent means ± SEM ( n > 15 per group). * P < 0.05; one-way ANOVA, Tukey’s multiple comparisons test. DMSO, dimethyl sulfoxide. ( B ) Requirement of ripk3 for the β cell loss. Data represent means ± SEM ( n > 10 per group). * P < 0.05; one-way ANOVA, Tukey’s multiple comparisons test. ( C ) Maintenance of whole-body glucose content in ripk3 −/− , zMIR larvae after three sessions of overnutrition. Data represent means ± SEM ( n = 4 per group). * P < 0.05; one-way ANOVA, Tukey’s multiple comparisons test. ( D ) Punctate distribution of mutant RIPK3 in zMIR but not in non-zMIR β cells at hour 64. Scale bars, 5 μm. ( E ) Protective effect of β cell–specific inhibition of RIPK3 on β cells. Data represent means ± SEM ( n > 25 per group). * P < 0.05; one-way ANOVA, Tukey’s multiple comparisons test. ( F ) Effect of β cell–specific inhibition of RIPK3 on whole-body free glucose content. Data represent means ± SEM ( n = 4 per group). * P < 0.05; one-way ANOVA, Tukey’s multiple comparisons test. ( G ) GSK’872 prevents palmitate-impaired insulin secretion. Average insulin secretion per islet at 1 mM and 16.7 mM glucose from mouse islets treated with or without 0.5 mM palmitate in the presence or absence of 3 μM GSK’872. Data represent means ± SEM ( n = 3 per group). ** P ≤ 0.01 and *** P ≤ 0.001; two-way ANOVA, Tukey’s multiple comparisons test.

Article Snippet: Primary antibodies used were guinea pig anti-human insulin (1:500) (DAKO, A0564), mouse anti-glucagon (1:500) (Abcam, ab10988), rabbit anti-human RIPK3 (1:200) (Aviva Systems Biology), mouse anti-human Grp78 (1:100) (Invitrogen), rat anti-mouse F4/80 [CI:A3-1] (1:100) (Abcam, ab6640), and rabbit anti-IL1B (1:100; Novus, NB600-633).

Techniques: Mutagenesis, Inhibition

Journal: Science Advances

Article Title: RIPK3-mediated inflammation is a conserved β cell response to ER stress

doi: 10.1126/sciadv.abd7272

Figure Lengend Snippet: ( A ) Western blot analysis of the p-RIPK3 levels in MIN6 cells treated with different stress conditions. Data represent means ± SEM ( n = 3 per group). *** P < 0.001; one-way ANOVA, Tukey’s multiple comparisons test. ( B ) Immunofluorescence analysis of RIPK3 distribution in MIN6 cells treated with different stress conditions. F-actin stain outlines individual cells. DNA stain labels nuclei. Scale bars, 10 μm. ( C ) Effects of Nec-1 and GSK’872 on thapsigargin- and GP-induced increase in p-RIPK3. Data represent means ± SEM ( n = 3 per group). * P < 0.05, ** P < 0.01, and *** P < 0.001; one-way ANOVA, Tukey’s multiple comparisons test. ( D ) Effects of Nec-1 and GSK’872 on the thapsigargin- and GP-induced increase in RIPK3 polymerization. Scale bars, 10 μm. ( E ) Effects of 4-PBA and TUDCA on the thapsigargin- and GP-induced increase in p-RIPK3. Data represent means ± SEM ( n = 3 per group). ** P < 0.01; one-way ANOVA, Tukey’s multiple comparisons test. ( F ) Effects of 4-PBA and TUDCA on the thapsigargin- and GP-induced increase in RIPK3 polymerization. Scale bars, 10 μm. ( G ) Effect of RIPK3 inhibition on the expression of proinflammatory genes in MIN6 cells. Bars labeled with a different letter are significantly different ( P < 0.05), while those sharing a letter are not significantly different ( P > 0.05). ( H ) Western blot analysis of p-p65 levels in MIN6 cells treated with different stress conditions. Data represent means ± SEM ( n = 3 per group). *** P < 0.001; one-way ANOVA, Tukey’s multiple comparisons test. ( I ) Effects of Nec-1 and GSK’872 on the thapsigargin- and GP-induced increase in p-p65. Data represent * P < 0.05 and means ± SEM ( n = 3 per group). ** P < 0.01; one-way ANOVA, Tukey’s multiple comparisons test. ( J ) Effects of 4-PBA and TUDCA on the thapsigargin- and GP-induced increase in p-p65. Data represent means ± SEM ( n = 3 per group). * P < 0.05 and ** P < 0.01; one-way ANOVA, Tukey’s multiple comparisons test.

Article Snippet: Primary antibodies used were guinea pig anti-human insulin (1:500) (DAKO, A0564), mouse anti-glucagon (1:500) (Abcam, ab10988), rabbit anti-human RIPK3 (1:200) (Aviva Systems Biology), mouse anti-human Grp78 (1:100) (Invitrogen), rat anti-mouse F4/80 [CI:A3-1] (1:100) (Abcam, ab6640), and rabbit anti-IL1B (1:100; Novus, NB600-633).

Techniques: Western Blot, Immunofluorescence, Staining, Inhibition, Expressing, Labeling

Journal: Science Advances

Article Title: RIPK3-mediated inflammation is a conserved β cell response to ER stress

doi: 10.1126/sciadv.abd7272

Figure Lengend Snippet: ( A ) qRT-PCR analysis of candidate macrophage attractants in the islets at hour 64. Data represent means ± SEM ( n = 4 per group). * P < 0.05; one-way ANOVA, Tukey’s multiple comparisons test. ( B ) qRT-PCR analysis of candidate macrophage attractants in the islets at hour 56. Data represent means ± SEM ( n = 4 per group). * P < 0.05; one-way ANOVA, Tukey’s multiple comparisons test. ( C ) RNAscope analysis of insulin, il1b , and il8a at hour 64 in the control and zMIR fish. Scale bars, 10 μm. ( D ) Representative IL1B and F4/80 immunofluorescence images of control and 1-week HFD mouse pancreas sections ( D′ ) and quantification of IL1B signal intensity in islet area. Scale bars, 20 μm. Inset scale bar, 5 μm. Data represent means ± SEM ( n = 3 per group). *** P ≤ 0.001; one-way ANOVA, Tukey’s multiple comparisons test. ( E ) RNAscope analysis of insulin, il1b , and il8a at hour 64 in the zMIR and zMIR; Tg(ins: RIPK3 D160N -GFP) fish. Scale bars, 10 μm. ( F and G ) Islet il1b expression and il8a by qRT-PCR. Data represent means ± SEM ( n = 4 per group). * P < 0.05; one-way ANOVA, Tukey’s multiple comparisons test. ( H ) qRT-PCR analysis of mouse islets treated with the control medium or medium containing palmitate (0.5 mM) in the presence or absence of 3 μM GSK for 48 hours. * P < 0.05. ( I ) Requirement of il1b for β cell loss. Data represent means ± SEM ( n > 9 per group). * P < 0.05; one-way ANOVA, Tukey’s multiple comparisons test. ( J ) Preservation of whole-body free glucose content in the il1b −/− zMIR larvae after three sessions of overnutrition. Data represent means ± SEM ( n = 4 per group). * P < 0.05; one-way ANOVA, Tukey’s multiple comparisons test.

Article Snippet: Primary antibodies used were guinea pig anti-human insulin (1:500) (DAKO, A0564), mouse anti-glucagon (1:500) (Abcam, ab10988), rabbit anti-human RIPK3 (1:200) (Aviva Systems Biology), mouse anti-human Grp78 (1:100) (Invitrogen), rat anti-mouse F4/80 [CI:A3-1] (1:100) (Abcam, ab6640), and rabbit anti-IL1B (1:100; Novus, NB600-633).

Techniques: Quantitative RT-PCR, RNAscope, Control, Immunofluorescence, Expressing, Preserving

Journal: Science Advances

Article Title: RIPK3-mediated inflammation is a conserved β cell response to ER stress

doi: 10.1126/sciadv.abd7272

Figure Lengend Snippet: ( A and B ) Representative images from live imaging. Live control (A) or zMIR (B) fish were imaged for macrophages (green) and β cells (red) every 30 min starting at hour 64. ( C to E ) Representative images from the fixed control larvae (C and C′) and zMIR larvae (D to E′). A single confocal slice (C, D, and E) and the corresponding projection (C′, D′, and E′) of the islet are shown. The inset in (E) shows a macrophage surrounding a β cell. Scale bars, 10 μm. ( F and F′ ) Requirement of Ripk3 for macrophage recruitment as shown by representative images (F) and distance between macrophage and nearest β cell (F′). Scale bars, 10 μm. Data represent means ± SEM ( n = 10 per group). * P < 0.05; one-way ANOVA, Tukey’s multiple comparisons test. Mø stands for macrophage. ( G and G′ ) Requirement of il1b for macrophage infiltration as shown by representative images (G) and by macrophage–to–β cell distance (G′) at hour 66. Scale bars, 10 μm. Data represent means ± SEM ( n = 10 per group). *** P < 0.001; one-way ANOVA, Tukey’s multiple comparisons test.

Article Snippet: Primary antibodies used were guinea pig anti-human insulin (1:500) (DAKO, A0564), mouse anti-glucagon (1:500) (Abcam, ab10988), rabbit anti-human RIPK3 (1:200) (Aviva Systems Biology), mouse anti-human Grp78 (1:100) (Invitrogen), rat anti-mouse F4/80 [CI:A3-1] (1:100) (Abcam, ab6640), and rabbit anti-IL1B (1:100; Novus, NB600-633).

Techniques: Imaging, Control

Journal: Science Advances

Article Title: RIPK3-mediated inflammation is a conserved β cell response to ER stress

doi: 10.1126/sciadv.abd7272

Figure Lengend Snippet: ( A ) Experimental design. ( B to B* ) Representative RIPK3 immunofluorescence images of human islet grafts from the PBS-treated (B) and DT-treated mice (B′) and (B*) and quantification of the percentage of β cells with the RIPK3 activation (B*). Data represent means ± SEM ( n = 4 per group). *** P < 0.001; one-way ANOVA, Tukey’s multiple comparisons test. Scale bars, 20 μm. Inset scale bar, 5 μm. ( C to C* ) Representative RIPK3 and GRP78 immunofluorescence images of human islet grafts from the PBS-treated (C) and DT-treated mice (C′) and quantification of the percentage of β cells with the RIPK3 activation (C*). Data represent means ± SEM ( n = 4 per group). *** P < 0.001; one-way ANOVA, Tukey’s multiple comparisons test. ( D to D* ) Representative GRP78 and p-p65 immunofluorescence images of human islet grafts from the PBS-treated (D) and DT-treated mice (D′) and quantification of the percentage of β cells with strong p-p65 signal (D*). Data represent means ± SEM ( n = 4 per group). *** P < 0.001; one-way ANOVA, Tukey’s multiple comparisons test. ( E to E* ) Representative RIPK3 and F4/80 immunofluorescence images of human islet grafts from the PBS-treated (E) and DT-treated mice (E′) and quantification of the total and RIPK3-positive macrophages (E*). Data represent means ± SEM ( n = 4 per group). *** P < 0.001; one-way ANOVA, Tukey’s multiple comparisons test. The transplants and human islets were part of a prior report , and sections of the graft were analyzed in the current study.

Article Snippet: Primary antibodies used were guinea pig anti-human insulin (1:500) (DAKO, A0564), mouse anti-glucagon (1:500) (Abcam, ab10988), rabbit anti-human RIPK3 (1:200) (Aviva Systems Biology), mouse anti-human Grp78 (1:100) (Invitrogen), rat anti-mouse F4/80 [CI:A3-1] (1:100) (Abcam, ab6640), and rabbit anti-IL1B (1:100; Novus, NB600-633).

Techniques: Immunofluorescence, Activation Assay