Journal: Biomolecules

Article Title: Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

doi: 10.3390/biom10030470

Figure Lengend Snippet: RIC3 antibodies used in this paper.

Article Snippet: Alomone Laboratories ANC-020 anti-RIC3 antibody showed a similar band pattern and recognized both splice variants of human and mouse RIC3, and rat RIC3 ( C).

Techniques:

Journal: Biomolecules

Article Title: Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

doi: 10.3390/biom10030470

Figure Lengend Snippet: RIC3 sequences across species showing antibody antigens and mutations.

Article Snippet: Alomone Laboratories ANC-020 anti-RIC3 antibody showed a similar band pattern and recognized both splice variants of human and mouse RIC3, and rat RIC3 ( C).

Techniques:

Journal: Biomolecules

Article Title: Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

doi: 10.3390/biom10030470

Figure Lengend Snippet: Assessment of RIC3 antibodies by Western blot. ( A ) Anti-DDK shows RIC3 expression efficiency following cell transfections. DDK–tagged RIC3s showed as single bands around 40 kD with considerable differences among different species. Rat RIC3 was not tagged with DDK. ( B ) Thermo-Fisher anti-hRIC3 PA5-64196 (1:1000) recognizes both splice variants of human and mouse RIC3 (with multiple bands) and weakly stains rat RIC3, but not Xenopus. ( C ) Alomone Laboratories anti-RIC3 antibody (ANC-020, 1:1000) showed a similar but weaker pattern and recognized both splice variants of human and mouse RIC3 (with additional bands) and weakly rat RIC3. A high MW (~100 kD) band is non-specific due to its presence in the control. Numbers in parentheses refer to the amount of protein added to each well to account for differences in protein expression between transfections.

Article Snippet: Alomone Laboratories ANC-020 anti-RIC3 antibody showed a similar band pattern and recognized both splice variants of human and mouse RIC3, and rat RIC3 ( C).

Techniques: Western Blot, Expressing, Transfection

Journal: Biomolecules

Article Title: Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

doi: 10.3390/biom10030470

Figure Lengend Snippet: ( A ) Autoradiographic comparison of 125 I-αBGT binding between wild type and KO animal brain slices. Top row shows total binding for wild type (left), tmem35a KO (middle) and ric3 KO (right) brain sections. The bottom row shows corresponding non-specific binding. There was no specific binding in tmem35a KO, and significant loss of binding in specific brain structures in the ric3 KO brains (arrows). ( B ) Autoradiographic analysis of 125 I-αBGT binding using ImageJ. Significant loss of toxin binding was observed in the hippocampus and cortex of the ric3 KO compared to the corresponding structures in wild type (WT) animals (Specific binding is the difference between total binding and non-specific [NS] binding). The insets show typical sections and the areas used for analysis over two sections per condition (N = 8 areas per brain region, with a medial and lateral area for each brain side times two sections). This analysis was done on one experiment comparing one animal per condition since the two experiments performed so far were done using different batches of 125 I-αBGT with different specific activities and slightly different exposure times and are not easily comparable. Error bars represent standard deviations. *** p > 0.001, (** p < 0.01, * p < 0.05) by single factor ANOVA.

Article Snippet: Alomone Laboratories ANC-020 anti-RIC3 antibody showed a similar band pattern and recognized both splice variants of human and mouse RIC3, and rat RIC3 ( C).

Techniques: Binding Assay

Journal: Biomolecules

Article Title: Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

doi: 10.3390/biom10030470

Figure Lengend Snippet: The absence of NACHO in HEK cells has no effect on the ability of RIC3 to promote surface human α7nAChR expression, and the effects of the two chaperones are synergistic when expressed together. Binding assays in 24-well plates were performed as indicated in methods. Total cDNA in transfections was constant, with h chrna7 DNA (0.15 μg/well) that was equaled the sum of htmem35a and hric3 cDNA or RFP DNA (0.15 μg/well RFP DNA in transfection controls). The ratio of 3 parts h tmem35a cDNA to 1 part h ric3 cDNA (e.g., 0.11 µg h tmem35a and 0.04 µg h ric3 /well) produced the highest surface α7nAChR expression in HEK cells. In all 4 experiments, the combined effects were more than additive. In experiments where RIC3 or NACHO was the only chaperone, surface α7nAChR expression was comparable between these two chaperones as shown.

Article Snippet: Alomone Laboratories ANC-020 anti-RIC3 antibody showed a similar band pattern and recognized both splice variants of human and mouse RIC3, and rat RIC3 ( C).

Techniques: Expressing, Binding Assay, Transfection, Produced

Journal: Cell reports

Article Title: Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery

doi: 10.1016/j.celrep.2023.112807

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-Cdc37 , StressMarq Biosciences , Cat# SPC-142; RRID:AB_2570605.

Techniques: Virus, Recombinant, Proximity Ligation Assay, Software

Journal: Stem Cells International

Article Title: Mesenchymal Stem Cells Inhibit Epithelial-to-Mesenchymal Transition by Modulating the IRE1 α Branch of the Endoplasmic Reticulum Stress Response

doi: 10.1155/2023/4483776

Figure Lengend Snippet: Suppression of ER stress contributed to MSC-mediated amelioration of EMT in A549 cells. (a) A549 cells were treated with 100 μ M TUDCA for different times. The protein expression levels of BiP, ATF6, ATF4, XBP-1s and XBP-1u were measured using western blotting and quantified using densitometry in ImageJ software ( n = 3, one-way ANOVA with Duncan's post hoc test). (b) A549 cells were treated with 10 ng/ml TGF- β 1 in the presence or absence of TUDCA for 72 hr. The protein expression levels of BiP, ATF6, ATF4, XBP-1s and XBP-1u were measured using western blotting and quantified using densitometry in ImageJ software ( n = 4, one-way ANOVA with Duncan's post hoc test). (c) A549 cells were treated with 100 μ M TUDCA for different times. The protein expression levels of E-cadherin and vimentin were measured using western blotting and quantified using densitometry in ImageJ software ( n = 3, one-way ANOVA with Duncan's post hoc test). (d) A549 cells were treated with 10 ng/ml TGF- β 1 in the presence or absence of TUDCA for 72 hr. The protein expression levels of E-cadherin and vimentin were measured using western blotting and quantified using densitometry in ImageJ software ( n = 4, one-way ANOVA with Duncan's post hoc test). The data are shown as the means ± SEMs ( ∗∗ P < 0.01, ∗ P < 0.05 vs. the control group; ## P < 0.05, # P < 0.05 vs. the TGF- β 1 group).

Article Snippet: A549 cells were treated with TGF- β 1 (10 ng/ml, 100-21, PeproTech, Rocky Hill, NJ, USA) for 72 hr with or without pretreatment with the ER stress inhibitor TUDCA (100 μ M, abs816166; Absin, Shanghai, China) or IRE1 α -specific inhibitor 4 μ 8c (10 μ M, T6363, Topscience, Shanghai, China) for 1 hr.

Techniques: Expressing, Western Blot, Software, Control

Journal: Journal of Advanced Research

Article Title: Bacteroides uniformis ameliorates pro-inflammatory diet-exacerbated colitis by targeting endoplasmic reticulum stress-mediated ferroptosis

doi: 10.1016/j.jare.2024.11.025

Figure Lengend Snippet: Pro-inflammatory diet inhibits endoplasmic reticulum stress signaling involving ATF4/HSPA5 pathway. (A) RNA-seq analysis on principal component analysis of all genes to evaluate the differences among CN group (in black, n = 4) and PI group (in red, n = 5). (B) Number of differentially expressed colonic genes between two groups. (C) Heatmap of gene set variation analysis showing several downregulated genes in PI group belonging to heat shock protein 70 family. (D) Quantitative RT–PCR analysis showing the down-regulation of Hspa1b , Hspa1a , Hspa5 and Hsph1 after pro-inflammatory diet treatment. (E) Comparisons of the relative mRNA level of Perk , Atf4 , Atf6 and Chop between mice treated with or without pro-inflammatory diet. (F) Heatmap on the expression of Perk , Atf4 , Hspa5 and GPX4 between ND group and PD group. (G-I) Western blotting and immunohistochemical results demonstrating decreased expression of ATF4 and HSPA5 in PD group. Corresponding animal experiments had been repeated for 3 or more times. Data are represented as the mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Upon reaching 70–80 % confluence, the cell were treated with HSPA5 inhibitor (1 uM, HY-100437, MCE, Shanghai, China), HSPA5 recombined protein (10 ng / mL, HY-P71742, MCE, Shanghai, China) or PERK inhibitor (1uM or 5uM, GSK2606414, MCE, Shanghai, China) for two days and were then harvested for RT-qPCR and Western blot analysis.

Techniques: RNA Sequencing, Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemical staining

Journal: Journal of Advanced Research

Article Title: Bacteroides uniformis ameliorates pro-inflammatory diet-exacerbated colitis by targeting endoplasmic reticulum stress-mediated ferroptosis

doi: 10.1016/j.jare.2024.11.025

Figure Lengend Snippet: The involvement of ferroptosis in intestinal inflammation of CD patients with pro-inflammatory diet pattern. (A-C) Results from Dataset Accession number GSE6731 showing the downregulation of GPX4 and the upregulation of HSPA5 and ATF4 in the disease affected colon from patients with IBD. CD-un, disease unaffected tissues from CD patients; CD-aff, disease affected tissues from CD patients; UC-un, disease unaffected tissues from UC patients; UC-aff, disease affected tissues from UC patients. (D-F) Relative expression of PERK , ATF4 , HSPA5 and GPX4 in colon from CD patients of our hospital in mRNA and protein level. HC, health control group; CD, patients with CD group; (G) The DII scores of 32CD patients included in this study were compared between High-DII group (patients with higher DII group) and Low-DII group (patients with lower DII group). (H) Representative immunohistochemical staining of GPX4 and HSPA5 in CD colon tissues from High-DII group and Low-DII group. (I-J) Quantification of GPX4 and HSPA5 expression using integrated optical density/specimen area (IOD/area). Data are represented as the mean ± SD. ns, no significance.

Article Snippet: Upon reaching 70–80 % confluence, the cell were treated with HSPA5 inhibitor (1 uM, HY-100437, MCE, Shanghai, China), HSPA5 recombined protein (10 ng / mL, HY-P71742, MCE, Shanghai, China) or PERK inhibitor (1uM or 5uM, GSK2606414, MCE, Shanghai, China) for two days and were then harvested for RT-qPCR and Western blot analysis.

Techniques: Expressing, Control, Immunohistochemical staining, Staining

Journal: Journal of Advanced Research

Article Title: Bacteroides uniformis ameliorates pro-inflammatory diet-exacerbated colitis by targeting endoplasmic reticulum stress-mediated ferroptosis

doi: 10.1016/j.jare.2024.11.025

Figure Lengend Snippet: Bacteroides uniformis ameliorates DSS-induced acute colitis. (A) Experimental design for antibiotics and DSS treatment, as well as the microbiota transplantation. BU group (oral administration of B. uniformis , n = 5), UA group (oral S. mutans , n = 5), and PBS group ( n = 5). (B) Culture of the fecal bacteria before and after antibiotic cocktails treatment using the blood agar plate. (C-D) Measurement of body weight lost and colon length at sacrifice of three groups. (E-F) H&E staining of colon section and the pathological score for each group (Scale bars: 625um). (G) Comparison of colonic MPO activity among three groups. (H-I) Detection of the relative mRNA level of Mucin-2 , tight juncture proteins ( Zo-1 and Cldn8 ) and inflammatory cytokines ( Tnfα , Il17a , Il6 , Il1β , Il12 , Il1α , Il10 and Tgfβ1 ) in colon. (J-L) Higher expression of GPX4, HSPA5 and ATF4 in both RNA and protein level after administrated with B. uniformis. Corresponding animal experiments had been repeated for 3 or more times. Data are represented as the mean ± SD.

Article Snippet: Upon reaching 70–80 % confluence, the cell were treated with HSPA5 inhibitor (1 uM, HY-100437, MCE, Shanghai, China), HSPA5 recombined protein (10 ng / mL, HY-P71742, MCE, Shanghai, China) or PERK inhibitor (1uM or 5uM, GSK2606414, MCE, Shanghai, China) for two days and were then harvested for RT-qPCR and Western blot analysis.

Techniques: Transplantation Assay, Bacteria, Staining, Comparison, Activity Assay, Expressing

Journal: Journal of Advanced Research

Article Title: Bacteroides uniformis ameliorates pro-inflammatory diet-exacerbated colitis by targeting endoplasmic reticulum stress-mediated ferroptosis

doi: 10.1016/j.jare.2024.11.025

Figure Lengend Snippet: Bacteroides uniformis ameliorates Pro-inflammatory diet-induced colitis in DSS model by reversing ferroptosis. (A) Experimental design for dietary and DSS treatment. Mice fed on the pro-inflammatory diet were divided into three groups: BU group (oral B. uniformis , n = 5), UA group (oral S. mutans , n = 5), PBS group ( n = 5). (B-D) Comparisons of body weight lost during DSS treatment, the colon length and the colonic MPO activity at sacrifice among three groups. (E-F) Quantitative RT–PCR detection of inflammatory cytokines ( Tnfα , Il17a , Il6 , Il1β , Il12 Il1α , Il10 and Tgfβ1 ), tight junction proteins ( Cldn1 , Cldn2 , Jam and Cldn14 ) and Mucin-2 expression in colon. (G-H) Representative images with H&E staining and the pathological score to estimate intestinal inflammation (Scale bars: 625um). (I-K) Measurement of Fe, GSH and MDA content in colon. (L) Relative mRNA expression of ferroptosis related genes ( Acsl4 , Tfr1 , Nox1 , Cox1 , Gpx4 , Fpn , Fth1 , Ftl , Tf , Ireb2 , Slc7a11 and Slc3a2 ). (M) Representative images of transmission electron microscopy for distal colon. Arrows head to mitochondria. (N-O) The recovery of GPX4, HSPA5, ATF4 and PERK expression in both RNA and protein level after administrated with B. uniformis. Corresponding animal experiments had been repeated for 3 or more times. Data are represented as the mean ± SD.

Article Snippet: Upon reaching 70–80 % confluence, the cell were treated with HSPA5 inhibitor (1 uM, HY-100437, MCE, Shanghai, China), HSPA5 recombined protein (10 ng / mL, HY-P71742, MCE, Shanghai, China) or PERK inhibitor (1uM or 5uM, GSK2606414, MCE, Shanghai, China) for two days and were then harvested for RT-qPCR and Western blot analysis.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Staining, Transmission Assay, Electron Microscopy

Journal: Journal of Advanced Research

Article Title: Bacteroides uniformis ameliorates pro-inflammatory diet-exacerbated colitis by targeting endoplasmic reticulum stress-mediated ferroptosis

doi: 10.1016/j.jare.2024.11.025

Figure Lengend Snippet: Co-cultured with Bacteroides uniformis inhibits ER stress pathway-mediated ferroptosis in vitro . (A-B) Improvement of cell viability in a concentration- and time-dependent pattern when co-culture with B. uniformis . (C-D) Decreased level of LDH level in cell coculture supernatant. Relative expression of PERK, ATF4, HSPA5 and GPX4 in RNA level (E, F) and in protein level (G H) after treated cells with B. uniformis . A, C, E and G showed the experimental results in Caco-2 cell. B, D, F and H showed the experimental results in NCM460 cell. Experiments had been repeated for 3 or more times. Data are represented as the mean ± SD.

Article Snippet: Upon reaching 70–80 % confluence, the cell were treated with HSPA5 inhibitor (1 uM, HY-100437, MCE, Shanghai, China), HSPA5 recombined protein (10 ng / mL, HY-P71742, MCE, Shanghai, China) or PERK inhibitor (1uM or 5uM, GSK2606414, MCE, Shanghai, China) for two days and were then harvested for RT-qPCR and Western blot analysis.

Techniques: Cell Culture, In Vitro, Concentration Assay, Co-Culture Assay, Expressing

Journal: Molecular Oncology

Article Title: The synthetic oleanane triterpenoid CDDO‐2P‐Im binds GRP78 / BiP to induce unfolded protein response‐mediated apoptosis in myeloma

doi: 10.1002/1878-0261.13447

Figure Lengend Snippet: CDDO‐2P‐Im binds GRP78 and activates the PERK and IRE1α branches of the UPR in a dose‐dependent manner. (A) Cell lysate of RPMI‐8226 was incubated with DMSO or CDDO‐2P‐Im for 30 min. Lysates were then subjected to 0.025 mg·mL −1 pronase for 30 min at 4 °C, followed by SDS/PAGE and western blot using antibodies against GRP78 and GAPDH. CDDO‐2P‐Im protects GRP78 from pronase cleavage compared with DMSO control. (B) Thermal shift assay was performed on recombinant GRP78 treated with control (DMSO) or CDDO‐2P‐Im. Relative fluorescent units (RFU) were measured by a SpectraMax Paradigm plate reader. The melting temperature (Tm) has a shift of −1.9 °C with the treatment of CDDO‐2P‐Im. (C, D) ARH‐77 and RPMI‐8226 cells were incubated with CDDO‐2P‐Im for 6 h. Cells were lysed and samples were prepared for SDS/PAGE. Western blot of UPR signaling proteins and downstream targets were performed. (E) 5T33 tumors were extracted from mice 15 days after tumor injection. Mice were treated once with either vehicle or 24 mg·kg −1 CDDO‐2P‐Im for 12 h prior to extraction. Samples were immunoblotted for UPR proteins. (F) RNA‐Sequencing data for ATF6 regulated genes showed no differences between Control, 0.1 μ m CDDO‐2P‐Im, and 0.4 μ m CDDO‐2P‐Im in RPMI‐8226 cells. (G, H) Western blot of ATF6 in ARH‐77 and RPMI‐8226 show no changes at 6 h with the treatment of CDDO‐2P‐Im. Values were given as mean ± SD. Fragments Per Kilobase of transcript per Million mapped reads is abbreviated as FPKM. Western blot and DARTS data are representatives of at least three independent experiments for ARH‐77 cells and RPMI‐8226 cells. Thermal shift assay was performed three independent times and representative data was shown. Western blot of 5T33 tumors was performed once. RNA‐Sequencing was performed once. Student t ‐tests were performed to calculate P value. 2P‐Im, CDDO‐2P‐Im; CDDO, CDDO‐2P‐Im.

Article Snippet: 10 μ m of recombinant GRP78 protein (PROTP11021; Boster Biological Technology, Pleasanton, CA, USA) was incubated with 150 μ m 2P‐Im controlled with 1% DMSO, and thermal shift assay was performed as previously described [ ].

Techniques: Incubation, SDS Page, Western Blot, Control, Thermal Shift Assay, Recombinant, Injection, Extraction, RNA Sequencing

Journal: Molecular Oncology

Article Title: The synthetic oleanane triterpenoid CDDO‐2P‐Im binds GRP78 / BiP to induce unfolded protein response‐mediated apoptosis in myeloma

doi: 10.1002/1878-0261.13447

Figure Lengend Snippet: Inhibition of the PERK‐ATF4‐CHOP arm of the UPR partially rescues CDDO‐2P‐Im‐induced apoptosis. (A) Cell lysate of WT and PERK KO RPMI‐8226 were probed for PERK protein by western blot. (B) WT and DDIT3 (CHOP) KO RPMI‐8226 were incubated with DMSO or CDDO‐2P‐Im for 6 h and lysed for protein analysis. Western blot of CHOP was performed to investigate the knockout of CHOP protein. (C) Cells were preincubated with DMSO or CDDO‐2P‐Im for 24 h and evaluated for cell viability by CellTiter‐Glo™. (D) Cells were preincubated with DMSO or 0.25 μ m ISRIB for 3 h before treating with DMSO or CDDO‐2P‐Im for an additional 16 h. Cells were then measured for cell viability by CellTiter‐Glo™. Controls were cells that were not treated with CDDO‐2P‐im but were treated with DMSO or ISRIB where appropriate. (E–H) PERK KO and WT RPMI‐8226 cells or ARH‐77 cells pretreated with ISRIB for 3 h were incubated with DMSO control or 0.4 μ m CDDO‐2P‐Im for 6 h. (E, F) Cells were extracted for RNA and qRT‐PCR was performed to investigate changes in the UPR. (G, H) In another round of experiments, cells were also extracted for protein to confirm such changes in the UPR. Values were given as mean ± SD. Student t ‐tests were performed to calculate P value. * P < 0.05 and ** P < 0.01, compared with control. Cell viability data are representative of three independent experiments. Western blots and qRT‐PCR data are representatives of two independent experiments. (I) We provide a working model of the actions of CDDO‐2P‐Im in cancer cells. At low concentrations (panel A), 2P‐Im binds and inhibits KEAP1, an adaptor protein for ubiquitin ligase that negatively regulates Nrf2 levels. Nrf2 will translocate to the nucleus and dimerize with small Maf proteins (sMaf) to activate the transcription of Nrf2 target genes. At higher concentrations (panel B), 2P‐Im binds GRP78/BiP, resulting in the activation of the UPR. GRP78 dissociates from its binding partners leading to the phosphorylation of PERK and IRE1α and activation of these branches of the UPR. The transcription factors, CHOP and XBP1, will cause UPR‐associated gene expression changes which when prolonged will lead to apoptosis. Interestingly, XBP1 is known to increase the expression of HRD1, a negative regulator of Nrf2, and independent of KEAP1. 2P/2P‐Im, CDDO‐2P‐Im; ISRIB, integrated stress response inhibitor; WT, wild‐type; KO, knockout.

Article Snippet: 10 μ m of recombinant GRP78 protein (PROTP11021; Boster Biological Technology, Pleasanton, CA, USA) was incubated with 150 μ m 2P‐Im controlled with 1% DMSO, and thermal shift assay was performed as previously described [ ].

Techniques: Inhibition, Western Blot, Incubation, Knock-Out, Control, Quantitative RT-PCR, Ubiquitin Proteomics, Activation Assay, Binding Assay, Phospho-proteomics, Gene Expression, Expressing

Journal: Molecular Cancer

Article Title: COSMC knockdown mediated aberrant O-glycosylation promotes oncogenic properties in pancreatic cancer

doi: 10.1186/s12943-015-0386-1

Figure Lengend Snippet: Expression of aberrant O-glycans in pancreatic cancer. a Biosynthesis of Tn antigen, sTn antigen and Core1 and 3 structures. Tn antigen is composed of an O-glycosidic linked N -acetylgalactosamine (GalNAc) to the –OH group of serine/threonine (S/T). Tn antigen is either processed by core 1 T-synthase (C1GalT1) and its chaperone (COSMC), which transfers a galactose (Gal) to GalNAc-serine/threonine to form the T antigen also referred as core 1 structure or processed by transfer of a N -acetylglucosamine (GlcNAc) to form the core 3 structure. Tn antigen can also be modified by addition of a sialic acid (NeuAc). b Differential expression of Tn antigen in pancreatic carcinoma cell lines. Eight different PDAC cell lines were available for analysis. Western and Far-Western blot analysis of total cell lysates was performed using the Tn antigen specific antibody MA1-80055. Detection of HSC70 served as loading control. Jurkat cells were used as positive control for Tn antigen expression. c Expression of Tn antigen and aberrant O-glycans in COSMC knockdown cells. Western blot analysis showed a strong expression of aberrant O-glycans as well as Tn antigen in Panc-1 COSMC knockdown cells compared to control cells. Sialyl-Tn and Tn antibodies were used as well as lectins such as VVL ( Vicia villosa lectin) and WFL ( Wisteria floribunda lectin)

Article Snippet: Tn antigen antibody (MA1-80055, clone BRIC111, Thermo Fischer), sTn antigen antibody (ABIN356328, clone STN218, antibodies online) and COSMC antibody (19254-1-AP, Proteintech) were diluted 1:250 in TBS-T. HSC70 (sc-7298), GAPDH (sc-32233,) Nucleolin (sc-8031), GRP78 (sc-1051), Alpha Enolase (sc-7455) and Annexin II (sc-9061) antibodies were purchased from Santa Cruz and diluted 1:1000.

Techniques: Expressing, Modification, Quantitative Proteomics, Western Blot, Far Western Blot, Control, Positive Control, Knockdown

Journal: Molecular Cancer

Article Title: COSMC knockdown mediated aberrant O-glycosylation promotes oncogenic properties in pancreatic cancer

doi: 10.1186/s12943-015-0386-1

Figure Lengend Snippet: MS identified Nucleolin, GRP-78, α-Enolase and Annexin A2 display O-GalNAc glycosylation. a left : Tn antigen is expressed on Nucleolin, GRP-78, α-Enolase and Annexin A2. Immunoprecipitation and western blot analysis were performed using VVL lectin for Tn antigen precipitation and Nucleolin, GRP-78, α-Enolase and Annexin A2 antibodies for detection of specific protein signals. a right : Protein expression levels of the identified proteins in total cell lysates of COSMC knockdown as well as control cells remained unchanged, except for Annexin 2, which showed a decrease in Panc-1 COSMC knockdown cells. b Vice versa, immunoprecipitation using specific antibodies and detection with VVL lectin was performed as well. c Immunocytochemistry of Panc-1 COSMC knockdown cells and control cells. Left : Aberrant O-glycans, including Tn antigen, were detected using VVL-FITC conjugated lectin ( green ). Middle left : Nucleolin was detected ( red ) using an anti-Nucleolin antibody (ab13541). Middle right: Nuclei were stained with DAPI ( blue ). Right : Overlay of Tn antigen and Nucleolin immunocytochemistry. d Nucleolin, GRP-78, Enolase and Annexin detection in the cell membrane protein fraction. Her2 antibody was used as marker for membrane fraction and GAPDH antibody was used as marker for cytoplasmic proteins

Article Snippet: Tn antigen antibody (MA1-80055, clone BRIC111, Thermo Fischer), sTn antigen antibody (ABIN356328, clone STN218, antibodies online) and COSMC antibody (19254-1-AP, Proteintech) were diluted 1:250 in TBS-T. HSC70 (sc-7298), GAPDH (sc-32233,) Nucleolin (sc-8031), GRP78 (sc-1051), Alpha Enolase (sc-7455) and Annexin II (sc-9061) antibodies were purchased from Santa Cruz and diluted 1:1000.

Techniques: Glycoproteomics, Immunoprecipitation, Western Blot, Expressing, Knockdown, Control, Immunocytochemistry, Staining, Membrane, Marker