Journal: Cell Stress & Chaperones

Article Title: Drug-induced keratin 9 interaction with Hsp70 in bladder cancer cells

doi: 10.1007/s12192-018-0913-2

Figure Lengend Snippet: Hsp70 and IgG antibodies were linked to Dynabeads, and T24 bladder cancer cell lysates were immunoprecipitated. SDS-PAGE, followed by silver staining, was performed and the band at the arrow was sliced from the gel and analyzed by LC-MS/MS. The unknown band was determined to be keratin 9. a Hsp70-Dynabead-linked Immunoprecipitation of T24 lysates in the absence of VER155008 (VER-) and presence of VER155008 (VER+). Lane C is an IgG-Dynabead-linked immunoprecipitate of a DMSO-treated T24 lysate (IgG control). Molecular weight markers (MWM) of 25, 50, and 75 represent molecular weights in kilodaltons. b bold black and red letters represent keratin 9 amino acid fragments identified from the excised band by LC-MS/MS. Red letters identify the amino acids found in the filament super family

Article Snippet: The protocol was followed according to the manufacturer’s instructions, beginning with the addition of 10 μL of Hsp70 antibody (StressMarq) or 10 μL of mouse IgG (Santa Cruz Biotechnology) to create the Co-IP bead complex.

Techniques: Immunoprecipitation, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Molecular Weight

Journal: Cell Stress & Chaperones

Article Title: Drug-induced keratin 9 interaction with Hsp70 in bladder cancer cells

doi: 10.1007/s12192-018-0913-2

Figure Lengend Snippet: Western blot analysis of human bladder cancer cell lysates (T24) treated with DMSO, the Hsp90 inhibitor STA9090, the Hsp70 inhibitors VER155008, and MAL3-101 or combinations of the various inhibitors. The blot was probed with a keratin 9 mouse monoclonal antibody

Article Snippet: The protocol was followed according to the manufacturer’s instructions, beginning with the addition of 10 μL of Hsp70 antibody (StressMarq) or 10 μL of mouse IgG (Santa Cruz Biotechnology) to create the Co-IP bead complex.

Techniques: Western Blot

Journal: Cell Stress & Chaperones

Article Title: Drug-induced keratin 9 interaction with Hsp70 in bladder cancer cells

doi: 10.1007/s12192-018-0913-2

Figure Lengend Snippet: Immunostaining using keratin 9 and Hsp70 mouse monoclonal antibodies and visualized by confocal microscopy. T24 human bladder cancer cells were processed as described in “ .” The negative controls without primary antibodies ( a , keratin 9) and ( c , Hsp70) show slight background staining resulting from autofluorescence of the secondary antibody. The nuclei were stained with DAPI. Perinuclear distribution of keratin 9 ( b ) and punctate distribution of Hsp70 ( d )

Article Snippet: The protocol was followed according to the manufacturer’s instructions, beginning with the addition of 10 μL of Hsp70 antibody (StressMarq) or 10 μL of mouse IgG (Santa Cruz Biotechnology) to create the Co-IP bead complex.

Techniques: Immunostaining, Confocal Microscopy, Staining

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: 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