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