bip  (Cell Signaling Technology Inc)

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: Extracellular GRP78 induces drug resistance through binding to cell surface ROR1, Cripto-1, and PD-L1. A, five-day cellular viability of U118, U87, and DIPG13 glioma cells with and without extracellular GRP78 (5 μg/ml) and under treatment at different concentrations of doxorubicin was measured with a live-cell CCK-8 assay. B, pediatric GBM SF9402, adult GBM U87, cell membrane proteins were biotinylated and solubilized using a nondenaturing membrane isolation kit and GRP78-bound proteins were eluted and analyzed by SDS-PAGE and mass spectrometry analysis. C, PAGE analysis of cell surface GRP78-binding proteins. Mass spectrometry analysis determined the bands were ROR1, PD-L1, and Cripto. MW. Molecular Weight Standards in kDa, GRP78 ppt = Surface proteins bound to GRP78. D, flow cytometry analysis of ROR1 expression on pediatric GBM SF9402 (43.4%) and adult GBM U87 (61.7%) cells. E, extracellular domains of ROR1, Cripto-1, PD-L1, and CD-44 were tested for GRP78 binding using a direct ELISA method. Kd = ROR1

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Binding Assay, CCK-8 Assay, Membrane, Isolation, SDS Page, Mass Spectrometry, Molecular Weight, Flow Cytometry, Expressing, Direct ELISA, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: Cell surface GRP78 colocalizes with ROR1, Cripto, and PD-L1 on late-stage patient GBM tissues but not on normal brain cerebrum tissues. A, three brain tissue microarrays with patient core tissue samples of GBM and normal tissues (#T174T from Biomax.us.) were stained for GBM markers. B, one microarray was stained with 4′,6-diamidino-2-phenylindole (DAPI) ( blue -DNA), and antibodies to cell surface GRP78 ( green -FITC), ROR1 ( red -PE). The last column is an overlay of all three stains to show colocalization. Cores B2 (stage 4 GBM) and C7 (normal cerebrum) are pictured. C, a second microarray was stained with DAPI ( blue -DNA), and antibodies to cell surface GRP78 ( green -FITC), PD-L1 ( red -PE). The last column is an overlay of all three stains to show colocalization. Cores B6 (stage 4 GBM) and C5 (normal cerebrum) are pictured. D, a third microarray was stained with DAPI ( blue -DNA), and antibodies to cell surface GRP78 ( green -FITC), PD-L1 ( red -PE). The last column is an overlay of all three stains to show colocalization. Cores B6 (stage 4 GBM) and C5 (normal cerebrum) are pictured. Scale bars represent 400 μm (10×). All images are from the same magnification of (10×) 400 μm. GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78; PD-L1, programed death-ligand 1; ROR1, receptor tyrosine kinase–like orphan receptor-1.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Staining, Microarray

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: CBT300’s ROR1 kringle domain (Kr1) domain binds to GRP78 and inhibits tumor cell proliferation. A, secondary structure of ROR1 showing where GRP78 is estimated to bind to ROR1 kringle domain. B, binding of individual ROR1 Extra Cellular Domains (Ig, FZD, KRD) and other kringle GRP78 binding domains (Kr1, Kr1Fc, K5) to GRP78. C, in silico docking of K5 kringle domain or the ROR1 kringle(Kr1) domain with GRP78. The lowest free energy of binding is shown for each of the kringle domains to GRP78. ROR1 kringle (Kr1) shows strong binding with two salt bridges, six hydrogen bonds and 98 nonbonded contacts to GRP78. D, graphic depiction of CBT100 (K5Fc) or CBT300 (Kr1Fc). E, SDS-PAGE gels of CBT100 and CBT300 showing reduced (#1) and nonreduced (#2) lanes. For both SDS-PAGE gels, M = molecular weight standards, #1 = reduced CBT100 (K5Fc) or CBT300 (Kr1Fc) and #2 = nonreduced CBT100 (K5Fc) or CBT300 (Kr1Fc). Size-exclusion analysis of CBT300 showing > 99% purity after protein A purification. F, five-day cell viability assay of U87 GBM cells treated with various concentrations of K5, CDT300 (Kr1Fc) CBT200 (K5PEG), and CBT100 (K5Fc) in triplicate wells. G, cell viability in a 5-day assay of four pediatric DIPG stem cell lines and two pediatric GBM stem cell lines under treatment with various concentrations of CBT300. The number of live cells was measured with a CCK-8 reagent. All dose response curves were fitted with a 3-parameter curve fits calculating the IC50 value. CCK-8, Cell Counting Kit-8; GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78; PD-L1, programed death-ligand 1; ROR1, receptor tyrosine kinase–like orphan receptor-1; DIPG, diffuse intrinsic pontine glioma; CBT, Creative BioTherapeutics.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Binding Assay, In Silico, SDS Page, Molecular Weight, Purification, Viability Assay, CCK-8 Assay, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: CBT300’s anticancer efficacy increases cell death in 2D assays and regresses tumor brain tumor spheroids in 3D assays with patient derived adult and pediatric glioma stem cells. A, CBT300 significantly increased cell death of patient derived glioma stem cells (827) grown on laminin. Three replicates of 60 cell count each were used with trypan blue reagent to determine dead cells. B and C, cell viability assays with pediatric and adult glioma cells. Cells were tested in triplicate repeats with doxorubicin with or without GRP78 (5 μg/ml) and with or without CBT300 (0.1 nM). Extracellular GRP78 increases resistance to doxorubicin and CBT300 reverses this resistance in cell viability assays. D, Pediatric Diffuse Intrinsic Ponte Glioma stem cells were seeded at 10,000 cells per well in 96 U-bottom well ultra low attachment (ULA) plates and cultured as a spheroid for 14 days with either medium, doxorubicin (10 μM), CBT300 (550 nM), or CBT300 (550 nM) + doxorubicin (1 μM). Representative images of DIPG-50 and DIPG-24 spheroids are shown. E– H , pediatric and adult glioma cells were seeded in ULA plates in triplicate wells with the treatments listed. Pictures of spheroids were taken at various times, and the size of the spheroids was measured by Image J ( https://imagej.net ). Spheroid regression/growth curves points are a mean of four replicates with standard deviation shown. The final point of measurements with a representative spheroid picture was shown with treatment regime and percent inhibition compared to the Control. Standard p values (∗ p < 0.5, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001) with respect to control were calculated by two-tailed unpaired t test for final measurements. I , patient derived adult 827 glioma stem cells were plated at 30,000 cells per well in 96-flat well plates coated with laminin. Cells were treated with either PBS control or CBT300 (550 nM) for 7 days. Spheroids containing more than or equal to five cells were counted on the days shown. Percent inhibition of spheroid numbers was calculated at the final time point. Standard p value (∗∗∗ p < 0.001) with respect to control was calculated by two-tailed unpaired t test for final measurements. The data points are an average of 3–4 replicates. CCK-8, Cell Counting Kit-8; GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78; PD-L1, programed death-ligand 1; ROR1, receptor tyrosine kinase–like orphan receptor-1; DIPG, diffuse intrinsic pontine glioma; CBT, Creative BioTherapeutics.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Derivative Assay, Cell Characterization, Cell Culture, Standard Deviation, Inhibition, Control, Two Tailed Test, CCK-8 Assay, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: CBT300 reduces expression of cell surface GRP78, ROR1, Cripto, and PD-L1. A, pediatric SF9427 DIPG cells (50,000) were incubated with either media, media+extracellular GRP78 (5 μg/ml), or media+extracellular GRP78 (5 μg/ml)+CBT300 (100 nM) for 72 h. Cells were then stained with mAbs for cell surface GRP78(csGRP78 nonpermeabilized cells), ROR1, Cripto, and PD-L1. Flow cytometry analysis was used on a Guava PCA to determine surface expression of listed cell surface proteins. The average for the percent positive cells is reported as the mean with SD from three replicate assays. B, adult GBM cells (50,000) were incubated with either media, media+extracellular GRP78 (5 μg/ml), or media+extracellular GRP78 (5 μg/ml)+CBT300 (100 nM) for 72 h. Cells were then stained with mAbs for cell surface GRP78(csGRP78 nonpermeabilized cells), ROR1, Cripto, and PD-L1. Flow cytometry analysis was used on a Guava PCA to determine surface expression of listed cell surface proteins. The average for the percent positive cells is reported as the mean with SD from three replicate assays. Studies were performed with three independent replicates. The average mean and SD for each set and marker are shown. Significance was determined between groups by two-way ANOVA analysis with post hoc Tukey’s test using GraphPad Prism 10.1.2. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. CCK-8, Cell Counting Kit-8; GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78; PD-L1, programed death-ligand 1; ROR1, receptor tyrosine kinase–like orphan receptor-1; DIPG, diffuse intrinsic pontine glioma; CBT, Creative BioTherapeutics.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Expressing, Incubation, Staining, Flow Cytometry, Marker, CCK-8 Assay, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: CBT300 reduces expression of cell surface GRP78 leading to increased doxorubicin internalization in glioma cells. A, U87 cells (20,000) were seeded in quadruplicate wells of tissue coated 96-well plates with either media (negative control – no doxorubicin), media (positive control – doxorubicin (2 μm)), media+GRP78 (5 μg/ml), or media+GRP78 (5 μg/ml)+CBT300 (100 nM) for 48 h. Doxorubicin (2 μM) was then added to all the wells except for the negative control wells. 24 h after adding doxorubicin to the cells, cells were washed and flow cytometry analysis for red doxorubicin fluorescence was performed. B, U87 cells (3,000) were plated in four well chamber slides and allowed to attach overnight. Each chamber was treated with either media, media+GRP78 (5 μg/ml), or media+GRP78 (5 μg/ml)+CBT300 (100 nM) for 48 h. Doxorubicin (2 μM) was then added to each chamber and incubated for 24 h. Cells/chambers were then washed and stained with a mAb-FITC ( green ) to the C-terminal domain of GRP78, and 4′,6-diamidino-2-phenylindole ( blue ) to the DNA. Doxorubicin, which has a natural red fluorescence, was also measured for each treatment. A chart of three independent repeats was compiled for each marker, cell surface GRP78, and increase in doxorubicin internalized. Studies were performed with three independent replicates. The average mean and standard deviation for each set and marker are shown. Significance was determined between groups by two-way ANOVA analysis with post hoc Tukey’s test using GraphPad Prism 10.1.2. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. C, the proposed mechanism of action of CBT300’s inhibition of surface expressed GRP78 leading to tumor regression and apoptosis. Cell surface GRP78 promotes tumor growth through stabilization of checkpoint protein PD-L1, oncofetal proteins ROR1, and Cripto (tumor promotion). CBT300 and CBT200 remove the surface GRP78, eliminating its stabilization of PD-L1, ROR1, and Cripto which then induces tumor apoptosis and reducing drug and immune resistance (tumor apoptosis). GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78; PD-L1, programed death-ligand 1; ROR1, receptor tyrosine kinase–like orphan receptor-1; CBT, Creative BioTherapeutics.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Expressing, Negative Control, Positive Control, Flow Cytometry, Fluorescence, Incubation, Staining, Marker, Standard Deviation, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: Antiangiogenesis and GRP78 inhibitor K5 (kringle 5), regresses GBM tumors and significantly extends survival with durable complete regressions in an orthotopic glioma murine model. A, experimental setup of intracranial stereotactic injections with D54 adult GBM cells in the brains of nude mice and subsequent treatment with K5(3 X14-day osmotic mini pumps) and MRI analysis on days 28, 42, and 65 after tumor cell inoculation. B, MRI analysis of orthotopic adult GBM D54 tumors treated with and without K5. C, Kaplan–Meier curves showing survival of K5-treated mice versus controls. D, GBM D54 tumor volume at day 65 or at death as determined by MRI analysis. E–G, immunohistochemical analysis of GBM D54 tumor tissues were analyzed for vessel density (CD31) and apoptotic cells (TUNEL) at day 65. Five tissue samples were stained from each group and the average with SD were plotted. Survival curves in ( C ) were compared using log-rank test. Statistical analysis in ( D ) was performed using two-way ANOVA with post hoc Tukey’s test. Statistical analysis in ( E ) and ( F ) was performed using one-way ANOVA with post hoc Dunnett’s test. CCK-8, Cell Counting Kit-8; GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Immunohistochemical staining, TUNEL Assay, Staining, CCK-8 Assay, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: GRP78 inhibition by systemic dosing of CBT300 (Kr1Fc) improves survival in preclinical models of glioblastoma cancer. A, experimental setup of stereotactic intracranial injection of adult PDx CTG-2687-Luc cells in the brains of nude mice and subsequent treatment with CBT300 (Kr1Fc). B, tumor bioluminescence of treated mice at days 0 and 27 of Control and CBT300-treated mice. C, Kaplan–Meier survival curve showing a significant 60% survival of CBT300 (Kr1Fc)-treated mice versus controls upon intracranial injection of PDx GBM cells. D, individual mice tumor volume as determined by bioluminescence. Control treated mice in black lines . CBT300-treated mice in red lines. E, average tumor volume as determined by bioluminescence for the CBT300 and Control groups up to day 27. N = 5 mice per group. F, immunofluorescence staining of CTG-2687 tumors for CBT300 and Control treatments. The scale bar represents 400 μm (10×). G, statical analysis of Control and CBT300-treated PDx GBM tumors for tumor cell number (4′,6-diamidino-2-phenylindole), CBT300 (IgG1), cell surface GRP78, ROR1, and Cripto expression as determined by immunofluorescence. Three independent mice GBM tumor tissues were measured. H, statical analysis of Control and CBT300-treated PDx GBM tumors for tumor expression of ABCC1, ABCB1, AGCG2, PD-L1, and Ki-67. Three independent mice GBM tumor tissues were measured. I, circulation concentrations of CBT300 in CBT300 treated and Control mice blood on day 27 2 h after last dose. Statistical analysis was performed using Kaplan–Meier survival curves in (C) and (D-H) were compared with an unpaired t test analysis using a two-stage step-up method by Benjamini, Krieger, and Yekutieli. GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78; PD-L1, programed death-ligand 1; ROR1, receptor tyrosine kinase–like orphan receptor-1; DIPG, diffuse intrinsic pontine glioma; PDx, patient-derived xenograft; CBT, Creative BioTherapeutics.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Inhibition, Injection, Control, Immunofluorescence, Staining, Expressing, Derivative Assay

Journal: iScience

Article Title: RNF126 mediates fetal growth restriction via ubiquitination-dependent degradation of the MYH9/MYH10 complex

doi: 10.1016/j.isci.2026.116285

Figure Lengend Snippet: ER Stress mediates the effect of RNF126 on trophoblast function (A) Western blot confirms stable overexpression of Flag-RNF126 (F-RNF126) in BeWo cells. Representative blot is shown from three independent experiments. (B and C) Stable expression of RNF126 inhibited proliferation (B) and promoted apoptosis (C) in BeWo cells. Data are shown as representative images and quantitative analyses ( n = 3 independent experiments). Scale bars, 50 μm. (D and E) The GO and KEGG enrichment analysis plot of the differentially expressed genes (DEGs; |Log2 FoldChange|>0.58, p < 0.05) between two groups. (F) Gene set enrichment analysis of RNA-seq data shows the enrichment of unfolded-protein-response-signaling related genes in OE-RNF126 cells compared to OE-NC cells. (G and H) Representative western blots and quantification show increased GRP78/BiP, pPERK, and CHOP with RNF126 overexpression, and reduced levels upon RNF126 knockdown under TM- or TG-induced ER stress for 24h ( n = 3 independent experiments). (I and J) Representative images and quantification show that 24h treatment with 4-PBA (1 mM) reverses RNF126 overexpression-induced changes in cell proliferation (I) and apoptosis (J) ( n = 3 independent experiments). Scale bars, 50 μm. Data in B, C, H, I, and J are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, n.s. not significant.

Article Snippet: GRP78/Bip , HUABIO , Cat#HA722202, RRID: AB_3718306.

Techniques: Western Blot, Over Expression, Expressing, RNA Sequencing, Knockdown

Journal: Neoplasia (New York, N.Y.)

Article Title: PRAS40 activates the IRE1α-XBP-1-mediated unfolded protein response to exacerbate colorectal cancer by enhancing ST6Gal1-dependent α-2, 6 sialylation of GRP78

doi: 10.1016/j.neo.2026.101297

Figure Lengend Snippet: Interaction between PRAS40 and GRP78. (A) Prediction of PRAS40’s binding proteins involved in ER stress and UPR by overlapping the PRAS40-binding proteins determined by Co-IP-MS and the ER stress- and UPR-related factors overexpressed in TCGA-CRC samples. (B) The unique peptides of GRP78 enriched in PRAS40-bound precipitates were determined by MS. (C-F) Co-IP analyses in HEK-293T and HT29 cells transfected with empty vector or Flag-PRAS40 expression vector (C, E), Flag-GRP78 expression vector (D, F). (G) GST pull-down assays. (H) Immunofluorescence staining with anti-PRAS40 and anti-GRP78 antibodies in HCT116 cells. (I) Co-IP analyses in HEK-293T cells transfected with empty vector or expression vectors of Flag-PRAS40 deletion mutants. Scale bar, 10 μm.

Article Snippet: Antibodies were purchased for detection of PRAS40 (Cell Signaling); GRP78, XBP-1, PARP, IRE1, Flag, GST, ST6Gal1, α-tubulin, β-actin (Proteintech); and SNA (Vector Laboratories).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Expressing, Immunofluorescence, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: PRAS40 activates the IRE1α-XBP-1-mediated unfolded protein response to exacerbate colorectal cancer by enhancing ST6Gal1-dependent α-2, 6 sialylation of GRP78

doi: 10.1016/j.neo.2026.101297

Figure Lengend Snippet: Effects of PRAS40 on the N-glycosylation of GRP78. (A) ssGSEA for the correlation between PRAS40 and N-glycan biosynthesis in TCGA-CRC samples. (B) SNA blotting analyses of peri-cancer and cancer tissues from mouse CRC models. (C-D) SNA blotting analyses of the HCT116 cells transfected with empty vector or Flag-PRAS40 expression vector (C) and control or PRAS40 shRNA (D). (E-G) SNA-pull down-MS in the cells transfected with empty vector or Flag-PRAS40 expression vector. CBB staining (E), the unique peptides spectrum of GRP78 (F) and the LC-MS/MS of amino acid 50-60 of GRP78 (G). (H) The HCT116 cells transfected with empty vector or Flag-PRAS40 expression vector was treated with or without PNGase F (1 mU), followed by western blotting analyses. (I-J) The HCT116 cells transfected with empty vector or Flag-PRAS40 expression vector (I), or control or PRAS40 shRNA, followed by SNA blotting analyses (J).

Article Snippet: Antibodies were purchased for detection of PRAS40 (Cell Signaling); GRP78, XBP-1, PARP, IRE1, Flag, GST, ST6Gal1, α-tubulin, β-actin (Proteintech); and SNA (Vector Laboratories).

Techniques: Glycoproteomics, Transfection, Plasmid Preparation, Expressing, Control, shRNA, Staining, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: PRAS40 activates the IRE1α-XBP-1-mediated unfolded protein response to exacerbate colorectal cancer by enhancing ST6Gal1-dependent α-2, 6 sialylation of GRP78

doi: 10.1016/j.neo.2026.101297

Figure Lengend Snippet: Effects of the N-glycosylation at Asn59 on the function of GRP78 and the UPR. (A) Predicted N-glycosylation sites in GRP78 by NetNGlyc-1.0 ( https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/ ). (B) Molecular docking of the interaction between PRAS40 and GRP78. PRAS40 was shown in red, and GRP78 was shown in blue, respectively. Hydrogen bonds were indicated by yellow dashed lines, and the numbers represented the lengths of hydrogen bonds. (C) The solvation energy effects (∆iG) of the interaction between PRAS40 and the predicted residues of GRP78 ( https://www.ebi.ac.uk/msd-srv/prot_int/pistart.html ). (D) The cells deleted with GRP78 were overexpressed with shRNA-resistant wild type GRP78 or GRP78 N59Q , followed with SNA pull down assays. (E-J) The cells deleted with GRP78 was overexpressed with Flag-PRAS40 together with or without shRNA-resistant wild type GRP78 or GRP78 N59Q , were treated with or without Tg. Cell viability analyses (E-F), flow cytometry analyses and the quantification of 3 experiments (G-H), western blotting analyses (I) and PCR analysis (J).

Article Snippet: Antibodies were purchased for detection of PRAS40 (Cell Signaling); GRP78, XBP-1, PARP, IRE1, Flag, GST, ST6Gal1, α-tubulin, β-actin (Proteintech); and SNA (Vector Laboratories).

Techniques: Glycoproteomics, shRNA, Flow Cytometry, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: PRAS40 activates the IRE1α-XBP-1-mediated unfolded protein response to exacerbate colorectal cancer by enhancing ST6Gal1-dependent α-2, 6 sialylation of GRP78

doi: 10.1016/j.neo.2026.101297

Figure Lengend Snippet: Effects of ST6Gal1-dependent α-2, 6 sialylation of GRP78 on the UPR. (A) Molecular docking of the interaction between GRP78 and ST6Gal1. GRP78 was shown in blue, and ST6Gal1 was shown in green, respectively. Hydrogen bonds were indicated by yellow dashed lines, and the numbers represented the lengths of hydrogen bonds. (B) Immunofluorescence staining with anti-GRP78 and anti-ST6Gal1 antibodies in HCT116 cells. (C-D) Co-IP followed by western blotting analyses in PRAS40-overexpressed HCT116 cells with anti-ST6Gal1 (C) and anti-GRP78 antibodies (D), respectively. (E-K) The HCT116 cells deleted with ST6Gal1 and overexpressed with Flag-PRAS40, were treated with or without Tg. SNA-pull down assays (E), cell viability analyses (F-G), flow cytometry analyses and the quantification of 3 experiments (H-I), western blotting analyses (J) and PCR analysis (K). Data represent the mean ± SD. Scale bar, 10 μm. ** P < 0.01; *** P < 0.001.

Article Snippet: Antibodies were purchased for detection of PRAS40 (Cell Signaling); GRP78, XBP-1, PARP, IRE1, Flag, GST, ST6Gal1, α-tubulin, β-actin (Proteintech); and SNA (Vector Laboratories).

Techniques: Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Western Blot, Flow Cytometry

Journal: bioRxiv

Article Title: HSF1-mediated Proteostasis Decline Links Aging and Sleep Disruption

doi: 10.64898/2026.04.21.719983

Figure Lengend Snippet: (A) Schematic of the aging multi-omics dataset. Bulk RNA-seq and proteomics (whole-tissue lysate, WTL; low-solubility fraction, LSF) were measured across eight mouse tissues (aorta, brain, heart, kidney, liver, lung, muscle, and skin) at 6, 15, 24, and 30 months (n = 4 per age). (B) RNA-protein directional coupling in aging. For each tissue, concordance of aging direction (slope of expression vs. age) between RNA and protein layers was quantified using Fisher’s exact test. Heatmap color shows log 2 (odds ratio). Overlaid numbers denote the corresponding odds ratios, shown for significant and biologically meaningful associations (FDR < 0.05 and odds ratio ≥ 1.5). (C) Representative RNA-protein aging trajectories (6-30 months) across tissues. Z-scored expression of selected genes ( Hspa5 , Hsph1 , Cirbp , Rbm3 ) is shown for RNA, WTL, and LSF. Proteostasis genes decline, whereas cold-shock genes increase, with varying RNA-protein concordance across tissues and fractions. (D) Reactome pathways enriched from genes showing concordant RNA-protein decline in aging and aligned with sleep restriction (SR). For each tissue, genes were selected based on consistent directionality across RNA and protein layers and concordant response under SR. Pathways were ranked by enrichment significance (-log 10 FDR), and recurrent pathways across tissues are shown. These include HSF1-mediated proteostasis programs, highlighting conserved molecular signatures across tissues.

Article Snippet: Proteostasis-related genes, including Hspa5 and Hsph1 , exhibited coordinated decline across RNA and protein layers, whereas cold-shock-associated genes such as Cirbp and Rbm3 showed concordant increases.

Techniques: Biomarker Discovery, RNA Sequencing, Solubility, Expressing

Journal: Journal of Advanced Research

Article Title: Tankyrase activity is essential for asymmetric division and chromosome segregation in oocyte meiosis

doi: 10.1016/j.jare.2025.07.008

Figure Lengend Snippet: TNKS affects distributions of Formin2 and ER for chromosome migration in mouse oocytes. (A) Representative images of Formin2 in MI stage oocyte from the control and JW55 treatment groups. Green, Formin2. Cyan, DNA. Bar = 20 μm. (B) Relative intensity of Formin2 at spindle poles in oocytes from the control (n = 47) and JW55 treatment (n = 51) groups. ***, P < 0.001. (C) Band intensity analysis of Formin2 expression in the MI stage oocytes from control and JW55 treatment groups. *, P < 0.05. (D) Representative images of ER-tracker in MI stage oocyte from the control and JW55 treatment groups. Black, ER-tracker. Bar = 20 μm. (E) The percentage of abnormal ER distribution in oocytes from the control (n = 50) and JW55-treated (n = 56) groups. *, P < 0.05. (F) Relative intensity of ER-tracker in oocytes from the control (n = 50) and JW55-treated (n = 56) groups. ***, P < 0.001. (G) Western blot of GRP78 in MI stage oocytes from control and JW55 treatment groups. (H) Band intensity analysis of GRP78 in the control and JW55 treatment groups. *, P < 0.05. (I) Representative images of a relative position change between chromosomes and Formin2 in ATI stage oocytes from the control and JW55 treatment groups. Green, Formin2. Cyan, DNA. Bar = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Rabbit anti-α-Tubulin antibody (11224-1-AP), rabbit polyclonal anti-GAPDH antibody (10494-1-AP), rabbit anti-GRP78 antibody (11587-1-AP), rabbit anti-TNKS1 antibody (18030-1-AP), rabbit anti-Ran antibody (10469-1-AP), and rabbit anti-Rab11a antibody (67902-1-Ig), rabbit anti-Securin antibody (18040-1-AP), rabbit anti-CDC25C antibody (66912-1-Ig), rabbit anti-Formin2 antibody (11259-1-AP), rabbit anti-BubR1 (11504-2-AP) were from Proteintech.

Techniques: Migration, Control, Expressing, Western Blot