grp94 antibody h 10 sc 393402  (Santa Cruz Biotechnology)

Journal: Journal of Extracellular Biology

Article Title: Proteomic Profiling of Bacterial Extracellular Vesicles for Exploring Ovarian Cancer Biomarkers

doi: 10.1002/jex2.70073

Figure Lengend Snippet: Characterisation and mass spectrometry analysis of patient ascites EV. (A) Schematics of EV collection from ascites samples. (B) EVs were recovered from the ascites samples of 10 patients with ovarian cancer and five patients without cancer by size exclusion (SEC). Size distribution obtained by NTAs for isolated EVs derived from representative samples of ascites samples. (C) Transmission electron microscopy analysis of isolated cancer and non‐cancer ascites EVs. Scale bar = 100 nm. (D) Immunoblot analyses for CD9, CD81 and GRP94 of EV samples of cancer and non‐cancer ascites samples. Uncropped Western blotting data are shown in Figure . (E) Schematics of mass spectrometry analysis of patient ascites EV with human reference. (F) MS data with human reference was obtained for cancer and non‐cancer ascites EV. According to protein content–based EV characterisation from MISEV2023, EV‐associated proteins were categorised into 1a to 5b. Each protein is identified by its gene symbol. The data were converted to a log10 scale. (G) The heat map shows the protein expression obtained from the same MS data as in Figure (total = 2284 proteins). The data were converted to log2 fold change (log2FC). The details of the protein names are given in the Supplementary Information. (H) PCA of the MS data obtained in Figure . (I) Volcano plot analysis between Cancer and non‐cancer samples of MS data obtained in Figure . The p values for each protein were calculated using the Wald test in Limma.

Article Snippet: After blocking with Blocking One (Nacalai tesque) for 1 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: anti‐CD9 Antibody, clone MM2/57 CBL162 (Merck, Darmstadt, Germany), CD81 Antibody (B‐11) sc‐166029 (Santa Cruz Biotechnology, Dallas, TX, USA) and GRP94 Antibody (H‐10) sc‐393402 (Santa Cruz Biotechnology), which were diluted 1:100 in 10 % Blocking One/Tris‐buffered saline with 0.1% Tween 20 (TBST).

Techniques: Mass Spectrometry, Isolation, Derivative Assay, Transmission Assay, Electron Microscopy, Western Blot, Expressing

Journal: iScience

Article Title: EDEM1 regulates the insulin mRNA level by inhibiting the endoplasmic reticulum stress-induced IRE1/JNK/c-Jun pathway.

doi: 10.1016/j.isci.2023.107956

Figure Lengend Snippet: Figure 3. EDEM1 overexpression increases insulin mRNA and consequently leads to an increased level of properly processed and matured proinsulin (A) INS-1E-pLPCX and cells stably expressing EDEM1, EDEM2 and EDEM3 were grown in RPMI-1640 medium with 11.2mM glucose for 72h and insulin mRNA level was determined by RT PCR. (B) As in (A), but INS-1E-pLPCX cells, heterogeneous bulk cultures (EDEM1B) and two cell clones (EDEM1C1 and EDEM1C2) expressing different amount of EDEM1 protein were analyzed. (C) As in (A), but INS-1E-shCtrl and INS-1E-shEDEM1 were used. (D) INS-1E-pLPCX and INS-1E-EDEM1 cells were seeded for 72h, starved in glucose-free medium for 1h and then incubated in medium with 1.7mM and 16.7 mM glucose for 1h and insulin mRNA level was analyzed by RT-PCR. (E) INS-1E-pLPCX and INS-1E-EDEM1 cells were grown in RPMI 1640 medium with 11.2mM glucose for 72h before being subjected to the subcellular fractionation by differential centrifugation and PNS, cytosol and ISG fractions were analyzed by Western blot with anti-insulin and anti-calnexin antibodies. (F) ISGs were separated by ultracentrifugation and analyzed on a discontinuous Nycodenz gradient composed of six layers (4.4%, 6%, 8.8%,11,7%,18% and 23,4%). Fractions were separated by SDS-PAGE and characterized with markers for ER (GRP78, GRP94), ER-Golgi intermediate compartment (ERGIC) (ERGIC53), clathrin vesicles (clathrin), secretory granules (syntaxin 8, VAMP1/2, VAMP2, PC2). (G) Proteins bands from Western Blot were analyzed and quantified with ImageJ analysis software. Data are represented as mean G SEM.

Article Snippet: Antibodies Insulin B (C-12) Santa Cruz Biotechnology Cat# sc-377071, RRID:AB_2800506 Insulin (H-86) Santa Cruz Biotechnology Cat# sc-9168, RRID:AB_2126540 C-peptide [1H8] Abcam Cat# ab8297, RRID:AB_306447 EDEM1 SIGMA-ALDRICH Cat# E8406, RRID:AB_1078720 Beta-Actin Abcam Cat# ab8226, RRID:AB_306371 Glut2 (C-19) Santa Cruz Biotechnology Cat# sc-7580, RRID:AB_641066 PC2 (H-20) Santa Cruz Biotechnology Cat#sc-22891, RRID:AB_2251901 Vamp2 (3E5) Santa Cruz Biotechnology Cat# sc-69706, RRID:AB_2212614 Vamp 1/2 (3H3117) Santa Cruz Biotechnology Cat# sc-73249, RRID:AB_1129807 GRP78 (A-10) Santa Cruz Biotechnology Cat# sc-376768, RRID:AB_2819145 GRP94 (H-10) Santa Cruz Biotechnology Cat# sc-393402, RRID:AB_2892568

Techniques: Over Expression, Stable Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Incubation, Fractionation, Centrifugation, Western Blot, SDS Page, Software

Journal: iScience

Article Title: EDEM1 regulates the insulin mRNA level by inhibiting the endoplasmic reticulum stress-induced IRE1/JNK/c-Jun pathway

doi: 10.1016/j.isci.2023.107956

Figure Lengend Snippet: EDEM1 overexpression increases insulin mRNA and consequently leads to an increased level of properly processed and matured proinsulin (A) INS-1E-pLPCX and cells stably expressing EDEM1, EDEM2 and EDEM3 were grown in RPMI-1640 medium with 11.2mM glucose for 72h and insulin mRNA level was determined by RT PCR. (B) As in (A), but INS-1E-pLPCX cells, heterogeneous bulk cultures (EDEM1B) and two cell clones (EDEM1C1 and EDEM1C2) expressing different amount of EDEM1 protein were analyzed. (C) As in (A), but INS-1E-shCtrl and INS-1E-shEDEM1 were used. (D) INS-1E-pLPCX and INS-1E-EDEM1 cells were seeded for 72h, starved in glucose-free medium for 1h and then incubated in medium with 1.7mM and 16.7 mM glucose for 1h and insulin mRNA level was analyzed by RT-PCR. (E) INS-1E-pLPCX and INS-1E-EDEM1 cells were grown in RPMI 1640 medium with 11.2mM glucose for 72h before being subjected to the subcellular fractionation by differential centrifugation and PNS, cytosol and ISG fractions were analyzed by Western blot with anti-insulin and anti-calnexin antibodies. (F) ISGs were separated by ultracentrifugation and analyzed on a discontinuous Nycodenz gradient composed of six layers (4.4%, 6%, 8.8%,11,7%,18% and 23,4%). Fractions were separated by SDS-PAGE and characterized with markers for ER (GRP78, GRP94), ER-Golgi intermediate compartment (ERGIC) (ERGIC53), clathrin vesicles (clathrin), secretory granules (syntaxin 8, VAMP1/2, VAMP2, PC2). (G) Proteins bands from Western Blot were analyzed and quantified with ImageJ analysis software. Data are represented as mean ± SEM.

Article Snippet: GRP94 (H-10) , Santa Cruz Biotechnology , , Cat# sc-393402, RRID: AB_2892568.

Techniques: Over Expression, Stable Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Incubation, Fractionation, Centrifugation, Western Blot, SDS Page, Software

Journal: iScience

Article Title: EDEM1 regulates the insulin mRNA level by inhibiting the endoplasmic reticulum stress-induced IRE1/JNK/c-Jun pathway

doi: 10.1016/j.isci.2023.107956

Figure Lengend Snippet:

Article Snippet: GRP94 (H-10) , Santa Cruz Biotechnology , , Cat# sc-393402, RRID: AB_2892568.

Techniques: Transduction, Recombinant, Enzyme-linked Immunosorbent Assay, Western Blot, shRNA, Software