Journal: Thoracic Cancer

Article Title: microRNA ‐100 functions as a tumor suppressor in non‐small cell lung cancer via regulating epithelial‐mesenchymal transition and Wnt/β‐catenin by targeting HOXA1

doi: 10.1111/1759-7714.13459

Figure Lengend Snippet: miR‐100 inhibited NSCLC cell proliferation. ( a ) qRT‐PCR was used to measure miR‐100 expressions in NSCLC cells. ( b , c ) miR‐100 overexpression or inhibition was confirmed by qRT‐PCR. ( d , e ) MTT assays were performed to detect the functions of miR‐100 in NSCLC cell proliferation. * P < 0.05, ** P < 0.01, *** P < 0.001 (d: NC and miR‐100 mimics; e: NC and miR‐100 inhibitor).

Article Snippet: Human normal bronchial epithelium cell BEAS‐2B and NSCLC cells (NCI‐H460, NCI‐H1299, SPC‐A1 and A549) were purchased from ATCC and maintained in DMEM (Gibco; Thermo Fisher Scientifc, Inc., Waltham, MA, USA) which contained 10% FBS (Gibco; Thermo Fisher Scientifc, Inc.) in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Quantitative RT-PCR, Over Expression, Inhibition

Journal: Thoracic Cancer

Article Title: microRNA ‐100 functions as a tumor suppressor in non‐small cell lung cancer via regulating epithelial‐mesenchymal transition and Wnt/β‐catenin by targeting HOXA1

doi: 10.1111/1759-7714.13459

Figure Lengend Snippet: miR‐100 suppressed the cell invasion and migration abilities of NSCLC cells. ( a , b ) The impacts of miR‐100 restoration on NSCLC cell invasion and migration were determined using transwell assays. ( c , d ) Transwell assay was performed to determine invasion and migration capacities of miR‐100 suppressed NSCLC cells. ** P < 0.01, *** P < 0.001.

Article Snippet: Human normal bronchial epithelium cell BEAS‐2B and NSCLC cells (NCI‐H460, NCI‐H1299, SPC‐A1 and A549) were purchased from ATCC and maintained in DMEM (Gibco; Thermo Fisher Scientifc, Inc., Waltham, MA, USA) which contained 10% FBS (Gibco; Thermo Fisher Scientifc, Inc.) in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Migration, Transwell Assay

Journal: Thoracic Cancer

Article Title: microRNA ‐100 functions as a tumor suppressor in non‐small cell lung cancer via regulating epithelial‐mesenchymal transition and Wnt/β‐catenin by targeting HOXA1

doi: 10.1111/1759-7714.13459

Figure Lengend Snippet: HOXA1 was a direct target of miR‐100 in NSCLC cells. ( a )The WT and MUT binding sites of miR‐100 on HOXA1 3′‐UTR. ( b ) Dual‐luciferase reporter assay was used to confirm the association between miR‐100 and HOXA1 in NSCLC cells ( NC and miR‐100 mimics). ( c , d ) Regulatory effects of miR‐100 on HOXA1 expressions in NSCLC cells. * P < 0.05, ** P < 0.01.

Article Snippet: Human normal bronchial epithelium cell BEAS‐2B and NSCLC cells (NCI‐H460, NCI‐H1299, SPC‐A1 and A549) were purchased from ATCC and maintained in DMEM (Gibco; Thermo Fisher Scientifc, Inc., Waltham, MA, USA) which contained 10% FBS (Gibco; Thermo Fisher Scientifc, Inc.) in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Binding Assay, Luciferase, Reporter Assay

Journal: Biology Open

Article Title: Endoplasmic reticulum stress-induced cellular dysfunction and cell death in insulin-producing cells results in diabetes-like phenotypes in Drosophila

doi: 10.1242/bio.046524

Figure Lengend Snippet: Induction of UPR observed in wing imaginal discs and IPCs with ectopic Hsc70-3 DN expression. (A–B) Expression of Xbp1-GFP generated by ER stress-dependent splicing of xbp1*-GFP mRNA in wing imaginal discs. Phase contrast (A,B) and fluorescence (A′,B′) micrographs of wing imaginal discs. (A,A′) Control wing disc ( Bx>xbp1*-GFP ). (B,B′) Wing disc expressing a dominant-negative form of Hsc70-3 in the wing pouch region (arrow) ( Bx>hsc70-3 DN , xbp1*-GFP ). (C–E) Fluorescence micrograph of wing discs stained with DAPI (white). (C′–E′) Immunostaining of the wing discs with an anti-GRP78 antibody. (D″) Immunostaining of the wing disc with anti-HA antibody. (C,C′) Fluorescence micrograph of a control wing imaginal disc ( Bx-Gal4/+ ). (D–D″) Wing imaginal disc expressing control Hsc70-3 in the wing pouch region of the imaginal disc ( Bx>hsc70-3 ). (E,E′) Wing imaginal disc expressing a dominant-negative form of Hsc70-3 in the same region ( Bx>hsc70-3 DN ). Anti-GRP78 immunostaining is shown in white. Note that more intense immunofluorescence was observed exclusively in areas expressing Hsc70-3 DN , but not the control protein. (A–F) Relative intensity of anti-GRP78 immunostaining in wing imaginal discs. Immunofluorescence signal intensity in each wing imaginal disc with the control Hsc70-3 ( n =31) or Hsc70-3 DN ( n =25) expression was calculated and normalized to the control value, which was set as 1.0 ( Bx-Gal4/+ ) ( n =25; n.s., not significant, P >0.05; *** P <0.001, Student's t -tests). Error bars represent s.e.m. (G–I) Anti-GRP78 immunostaining of IPCs expressing GFPnls in brains from third-instar larvae. (G) Control IPCs ( ilp2>GFPnls ), (H) IPCs expressing the control Hsc70-3 ( ilp2>hsc70-3, GFPnls ), (I) IPCs expressing Hsc70-3 DN ( ilp2>hsc70-3 DN , GFPnls ). Anti-GRP78 immunostaining is colored in red (G–I; white in G′–I′). Nuclei of IPCs visualized by GFPnls expression are colored green (G–I; white in G″–I″). Arrows in H′ and H″ indicate positions of IPC cells. Note that remarkably higher immunostaining signal was observed in IPCs expressing Hsc70-3 DN , but not the control protein. (J) Relative intensities of anti-GRP78 immunostaining in larval IPCs. Immunofluorescence signal intensities in each IPC expressing Hsc70-3 ( n =25) or Hsc70-3 DN ( n =21) were calculated and normalized to the control value of 1.0 ( ilp2>GFPnls ) ( n =21, * P <0.05, *** P <0.001, Student's t -test). Error bars represent s.e.m. Scale bars: (A–E) 100 µm, (G–I) 50 µm.

Article Snippet: The following primary antibodies were used at the dilution described; rabbit anti-β-galactosidase (MP Biomedicals, #55976) at 1:1000, rabbit anti-GRP78 (Bip) (StressMarq Biosciences Inc., Cadboro Bay, Victoria, Canada) that could recognize Hsp70 family proteins including Hsc70-3 in Drosophila at 1:500, rabbit Cleaved Caspase-3 (Asp175) (#9661, Cell Signaling, Danvers, Massachusetts, USA) at 1:200 for larval brain immunostaining and at 1:150 for wing disc immunostaining, rabbit anti-Cleaved Drosophila Dcp-1 (Asp216) (Cell Signaling, antibody #9578) at 1:500, and rabbit anti-phospho-SAPK/JNK (pThr183, pTyr185) (Calbiochem, La Jolla, CA, USA) at 1:200.

Techniques: Expressing, Generated, Fluorescence, Dominant Negative Mutation, Staining, Immunostaining, Immunofluorescence

Journal: PLoS ONE

Article Title: The Proprotein Convertase Furin Contributes to Rhabdomyosarcoma Malignancy by Promoting Vascularization, Migration and Invasion

doi: 10.1371/journal.pone.0161396

Figure Lengend Snippet: A) mRNA levels of all nine proprotein convertases (PCs) were determined by qRT-PCR in 5 Ewing sarcoma, 8 osteosarcoma and 20 rhabdomyosarcoma (RMS) cell lines. Shown are levels relative to GAPDH expression. B) Protein levels of proform and mature furin were assessed by immunoblotting in 20 different RMS cell lines. C) Endogenous furin activity of selected RMS cell lines: Rh36, RD (eRMS), Rh3, Rh4, Rh30, Rh41 and RMS13 (aRMS). Furin activity deficient cells LoVo cells serve as negative control. Furin was captured from cell lysates on anti-furin antibody coated plates and furin activity was measured by addition of the fluorogenic substrate Boc-RVRR-AMC after 6h. Displayed are values normalized by background subtraction.

Article Snippet: Black FluoroNunc 96 well plates (MaxiSorp surface, Nunc, Thermo Scientific) were coated with goat anti human furin antibody (AF1503, R&D Systems) at 10 μg/ml in 50 mM Na 2 CO 3 , pH 9.6 (50 μl/ well) for 8h at RT, protected from light.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Activity Assay, Negative Control

Journal: Cell reports

Article Title: Activation of endoplasmic reticulum stress in premature aging via the inner nuclear membrane protein SUN2

doi: 10.1016/j.celrep.2023.112534

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Hsp40 , StressMarq Biosciences , Cat#SPC-100; RRID:AB_2261861.

Techniques: Produced, Marker, Recombinant, SYBR Green Assay, Transgenic Assay, Expressing, Knock-In, Negative Control, Real-time Polymerase Chain Reaction, Derivative Assay, Plasmid Preparation, Construct, Software, Imaging, Light Microscopy, Laser-Scanning Microscopy, Super-Resolution Microscopy, FACS

Journal: Molecular Cancer

Article Title: Down-regulation of miR-675-5p contributes to tumor progression and development by targeting pro-tumorigenic GPR55 in non-small cell lung cancer

doi: 10.1186/s12943-015-0342-0

Figure Lengend Snippet: Down-regulation of miR-675-5p is inversely associated with advanced stage and lymph node metastasis of NSCLC. (A) miR-675-5p expression level was significantly lower in NSCLC tissues than in their matched normal tissues. (B) Low-level expression of miR-675-5p was associated with high tumor stage of NSCLC (P < 0.05). (C) Low-level expression of miR-675-5p was related with lymph node metastasis of NSCLC (P < 0.05). ( D) miR-675-5p expression in NSCLC cell lines and normal human bronchial epithelial cell line (HBE). Expression levels of miR-675-5p were determined by qRT-PCR and normalized against an endogenous control (U6 RNA). Data were represented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: Six NSCLC cell lines (HTB-182, A549, SPC-A-1, H1299, 95-D, Ltep-a-2) were obtained from the American Type Culture Collection.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Molecular Cancer

Article Title: Down-regulation of miR-675-5p contributes to tumor progression and development by targeting pro-tumorigenic GPR55 in non-small cell lung cancer

doi: 10.1186/s12943-015-0342-0

Figure Lengend Snippet: Overexpression of miR-675-5p inhibited proliferation and colony formation of NSCLC cells. (A) the level of miR-675-5p in A549 and HTB-182 cells was significantly up-regulated after transfection with miR-675-precursor. (B) miR-675-5p reduced cell proliferation in NSCLC cells. Cell proliferation was determined using MTT assays. (C) miR-675-5p induced cell cycle arrest at the G1/S phase. (D) miR-675-5p suppressed colony formation compared with controls. The number of colonies were calculated and depicted by the ban graph. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector. *P < 0.05, **P < 0.01.

Article Snippet: Six NSCLC cell lines (HTB-182, A549, SPC-A-1, H1299, 95-D, Ltep-a-2) were obtained from the American Type Culture Collection.

Techniques: Over Expression, Transfection, Negative Control, Plasmid Preparation

Journal: Molecular Cancer

Article Title: Down-regulation of miR-675-5p contributes to tumor progression and development by targeting pro-tumorigenic GPR55 in non-small cell lung cancer

doi: 10.1186/s12943-015-0342-0

Figure Lengend Snippet: Requirement of GPR55 for miR-675-5p induced suppression of NSCLC cell proliferation, migration and invasion. (A) the level of miR-675-5p in the cells was significantly down-regulated after transfection with miR-675-5p inhibitor. (B) GPR55 was knockdowned in Ltep-a-2 miR-675-5p inhibition cells and analyzed by western blot analysis. Knockdown of GPR55 significantly inhibited proliferation (C) , inhibited colony formation (D) , induced cell cycle arrest at the G1/S phase (E) , inhibited migration and invasion (F) , and decreased the wound healing rate (G) in Ltep-a-2 cells. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGC FU-RNAi-NC-LV. *P < 0.05, P* < 0.01.

Article Snippet: Six NSCLC cell lines (HTB-182, A549, SPC-A-1, H1299, 95-D, Ltep-a-2) were obtained from the American Type Culture Collection.

Techniques: Migration, Transfection, Inhibition, Western Blot, Knockdown, Negative Control

Journal: PLoS ONE

Article Title: Engineering, and production of functionally active human Furin in N . benthamiana plant: In vivo post-translational processing of target proteins by Furin in plants

doi: 10.1371/journal.pone.0213438

Figure Lengend Snippet: ( A): Western blot analysis of human furin, produced in N . benthamiana plants . N . benthamiana leaf samples were harvested at 6 dpi. Samples for western blot analysis were prepared as described in Materials and Methods. Proteins on the blot were probed with a purified mouse anti-His tag antibody. 1- crude extract from non-infiltrated N . benthamiana ; 2- crude extract from N . benthamiana plant infiltrated with pEAQ-Furin (truncated)-His-KDEL construct. M: MagicMark XP Western Protein Standard. ( B): SDS-PAGE analysis of Ni-NTA column purified plant produced recombinant human furin. 5 μg Ni-NTA column purified protein was loaded into well. ( C ): Western blot analysis of different dilutions of Ni-NTA column purified, plant produced recombinant human furin, along with protein standards. Partially purified, plant produced furin was diluted 2.5, 5, 10 and 25-fold and different amount of plant produced furin, as indicated, was run on SDS-PAGE, followed by western blot. Plant produced His tagged furin protein band was detected using a purified mouse anti-His tag antibody. The concentration of furin in Ni-NTA column purified samples was quantified using the gene tools software, Syngene Bioimaging. Plant produced Endo H deglycosylated, purified PA83 protein (dPA83) was used as a protein standard. M1: color prestained protein standard (NEB); M2: MagicMark XP Western Protein Standard (ThermoFisher Scientific). ( D, E ): schematic representation of the full length (D) and truncated furin (E) structures. SP- Signal peptide; PP- Propeptide; SD- Subtilisin-like catalytic domain; BD- Homo B domain; CD- Cysteine rich domain; TM- Transmembrane domain; CD- Cytoplasmic domain.

Article Snippet: To test the effect of in vitro deglycosylation on furin cleavage activity, furin proteins that were co-expressed with Endo H and PNGase F were purified from plant tissue using the Ni-NTA column as described in Materials and Methods above. demonstrates a SDS-PAGE analysis of plant produced dPA83 incubated with plant produced or commercial furin (NEB).

Techniques: Western Blot, Produced, Purification, Construct, SDS Page, Recombinant, Concentration Assay, Software

Journal: PLoS ONE

Article Title: Engineering, and production of functionally active human Furin in N . benthamiana plant: In vivo post-translational processing of target proteins by Furin in plants

doi: 10.1371/journal.pone.0213438

Figure Lengend Snippet: SDS-PAGE CBB (A, C) and Western blot (B) analysis of dPA83, cleaved with plant produced or commercial human Furin. (A) : 5 μg dPA83 (deglycosylated PA83) samples were treated with different concentrations (0, 1, 5, 20, 25, 50 and 100 ng) of plant produced human furin and then 4.5 μg samples were run on SDS-PAGE. dPA83: deglycosylated PA83; pp hFurin: plant produced, Ni-NTA column purified furin. M1: color prestained protein standard (NEB). ( B) : 5 μg of deglycosylated PA83 samples were treated with different concentrations of plant produced human furin or 50 ng commercial (NEB) human furin as indicated, and then 100 ng PA samples were loaded into the gel. dPA83: deglycosylated PA83; pp hFurin: plant produced, Ni-NTA column purified furin. M2: MagicMark XP Western Protein Standard. (C) : 5 μg dPA83 (deglycosylated PA83) samples were treated with different concentrations (0, 1, 5, 20, 25, 50 and 100 ng) of commercial human furin (NEB) as indicated, and then 4.5 μg samples were run on SDS-PAGE. ( D) : Schematic representation of PA83 protein structure. PA63 and PA20 (a 20-kDa amino-terminal fragment) are cleavage products of PA83 by furin. M1: color prestained protein standard (NEB).

Article Snippet: To test the effect of in vitro deglycosylation on furin cleavage activity, furin proteins that were co-expressed with Endo H and PNGase F were purified from plant tissue using the Ni-NTA column as described in Materials and Methods above. demonstrates a SDS-PAGE analysis of plant produced dPA83 incubated with plant produced or commercial furin (NEB).

Techniques: SDS Page, Western Blot, Produced, Purification

Journal: PLoS ONE

Article Title: Engineering, and production of functionally active human Furin in N . benthamiana plant: In vivo post-translational processing of target proteins by Furin in plants

doi: 10.1371/journal.pone.0213438

Figure Lengend Snippet: ( A ): Lanes: 1- Co-expression of PA83 with PNGase F for the production of PNGase F deglycosylated PA83 protein; 2- Co-expression of PA83 with Endo H for the production of Endo H deglycosylated PA83 protein; 3-Expression of PA83 ( alone) for the production of glycosylated PA83 protein; 4- Co-expression of PA83 with furin and PNGase F for the production of furin cleaved and PNGase F deglycosylated PA83 protein; 4- Co-expression of PA83 with furin and Endo H for the production of furin cleaved and Endo H deglycosylated PA83 protein; 5- Co-expression of PA83 with furin for the production of furin cleaved and glycosylated PA83 protein. ( B ): WB analysis of N . benthamiana plant, infiltrated with PA83 and Endo H or infiltrated with PA83, Endo H and Furin constructs. 6-7-week-old N . benthamiana plant leaves, were infiltrated with the above constructs, were harvested and samples were processed for SDS-PAGE and western blot, as described in Materials and Methods. Boiled and un-boiled (raw) samples were diluted 5-fold and 10 μl from each sample was run on SDS-PAGE prior to western blotting. Proteins were probed with the purified anti-His tag antibody. The image was taken using a highly sensitive GeneGnome XRQ Chemiluminescence imaging system. An arrow indicates the protein bands corresponding to PA83 and PA63 and the formation of PA63 oligomers. M: MagicMark XP Western Protein Standard.

Article Snippet: To test the effect of in vitro deglycosylation on furin cleavage activity, furin proteins that were co-expressed with Endo H and PNGase F were purified from plant tissue using the Ni-NTA column as described in Materials and Methods above. demonstrates a SDS-PAGE analysis of plant produced dPA83 incubated with plant produced or commercial furin (NEB).

Techniques: Expressing, Construct, SDS Page, Western Blot, Purification, Imaging

Journal: PLoS ONE

Article Title: Engineering, and production of functionally active human Furin in N . benthamiana plant: In vivo post-translational processing of target proteins by Furin in plants

doi: 10.1371/journal.pone.0213438

Figure Lengend Snippet: ( A ): Western blot analysis of Ni-NTA purified plant produced furin variants, i.e. glycosylated Endo H or PNGase F in vivo deglycosylated, as indicated. gfurin: plant produced furin, expressed alone (glycosylated); dfurin (E): plant produced furin co-expressed with Endo H; dfurin (P): plant produced furin co-expressed with PNGase F. Furin protein bands were detected using the purified anti-His tag antibody. M1: MagicMark XP Western Protein Standard. ( B ) SDS-PAGE CBB: 5.0 μg plant produced dPA83 was incubated with different amount (1.0, 5.0, 25, 50, 100 ng) of furin, which was co-expressed with PNGase F and then 2.5 μg PA83 protein from each sample was loaded into each well. G- positive control, 5.0 μg plant produced dPA83 was incubated with 50 ng of plant produced furin (glycosylated) and then 2.5μg dPA83 was loaded into a well and run on a SDS-PAGE. C-negative control, plant produced dPA83 protein, not incubated with furin. M2-color prestained protein standard (NEB); S- BSA standard. ( C ): 5.0 μg plant produced dPA83was incubated with different amount (1.0, 5.0, 25, 50, 100 ng) of furin co-expressed with Endo H and then 2.5 μg PA83 protein from each sample was loaded into each well. C-negative control, plant produced dPA83 protein, not incubated with furin. M2-color prestained protein standard (NEB); S- BSA standard. (D): 5.0 μg plant produced PA83 (deglycosylated) was incubated with commercial human furin, which was previously in vitro deglycosylated with commercial Endo H (lane 2) or PNGase F (Lane 3). Lane 1, positive control, 5.0 μg plant produced dPA83 was incubated with 50 ng commercial human furin (NEB) and 2.5μg PA83 was loaded into a well. C-negative control, plant produced dPA83 protein, not incubated with commercial furin. M2-color prestained protein standard (NEB); S- BSA standard.

Article Snippet: To test the effect of in vitro deglycosylation on furin cleavage activity, furin proteins that were co-expressed with Endo H and PNGase F were purified from plant tissue using the Ni-NTA column as described in Materials and Methods above. demonstrates a SDS-PAGE analysis of plant produced dPA83 incubated with plant produced or commercial furin (NEB).

Techniques: Western Blot, Purification, Produced, In Vivo, SDS Page, Incubation, Positive Control, Negative Control, In Vitro

Journal: Cell Death Discovery

Article Title: The chaperone GRP94 interacts with the proprotein convertase furin and regulates TGF-beta maturation in human primary M2 macrophages

doi: 10.1038/s41420-025-02866-2

Figure Lengend Snippet: A Analysis by microscale thermophoresis of the specific binding between Nano-RED-labelled recombinant GRP94 (100 nM) and either recombinant furin (3.58 μM to 0.1 nM) or the negative control recombinant Hsp110 (2.33 μM to 0.07 nM). Changes in thermophoresis depending on ligand (furin or Hsp110) concentration from n = 3 experiments were plotted and expressed as ∆Fnorm (‰). B Staining and quantification of proximity ligation assays (Duolink™ PLA) of GRP94 and furin or the negative control Hsp110 in human PBMC-derived M2 macrophages ( n = 3 donors). Scale bars = 50 µm. The bar plot represents the mean number of spots for each cell analyzed +/− SEM. (**** p < 0.0001). C GRP94-furin co-immunoprecipitation in M2 macrophages. GRP94 was immunoprecipitated in M2 macrophages cell lysates using an anti-GRP94 antibody (IP:GRP94) or a non-relevant antibody (IgCT). Samples were analyzed by western-blot (representative images, n = 7).

Article Snippet: Specific binding between recombinant human furin (1503-SE, R&D Systems®, USA) and recombinant human GRP94 (RD172379100, Biovendor®, Czech Republic) was measured by MST assays.

Techniques: Microscale Thermophoresis, Binding Assay, Recombinant, Negative Control, Concentration Assay, Staining, Ligation, Derivative Assay, Immunoprecipitation, Western Blot

Journal: Cell Death Discovery

Article Title: The chaperone GRP94 interacts with the proprotein convertase furin and regulates TGF-beta maturation in human primary M2 macrophages

doi: 10.1038/s41420-025-02866-2

Figure Lengend Snippet: A Furin enzymatic activity analysis in cell lysates of MDA-MB-231 treated or not 24 h with GRP94 inhibitors, PU-WS13 12.5 or 25 µM or GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM ( n = 6). Fluorescence signal (expressed in relative fluorescence units, RFU) due to cleavage of the furin fluorogenic substrate, pERTKR-AMC, in cell lysates was recorded during 30 min. (**** p < 0.0001). Western- blot analysis of ( B ) active TGFβ secretion in supernatants (representative images, n = 7) and ( C ) pro-TGFβ expression in cell lysates (representative images, n = 5) from MDA-MB-231 treated or not 24 h with GRP94 inhibitors, PU-WS13 12.5 or 25 µM or GRP94 inhibitor-1 (GRP94inh1) 2.5 or 5 µM. (* p < 0.05; ** p < 0.01). D GRP94-furin co-immunoprecipitation in MDA-MB-231 cells. GRP94 was immunoprecipitated in MDA-MB-231 cell lysates using an anti-GRP94 antibody (IP:GRP94) or a non-relevant antibody (IgCT). Samples were analyzed by western-blot (representative images, n = 4). E Staining and quantification of proximity ligation assays (Duolink™ PLA) of GRP94 and furin or the negative control Hsp110 in MDA-MB-231 cells treated or not 24 h with GRP94 inhibitor-1 5 µM ( n = 3 experiments). Scale bars = 20 µm. The bar plot represents the mean number of spots for each cell analyzed +/− SEM. (**** p < 0.0001).

Article Snippet: Specific binding between recombinant human furin (1503-SE, R&D Systems®, USA) and recombinant human GRP94 (RD172379100, Biovendor®, Czech Republic) was measured by MST assays.

Techniques: Activity Assay, Fluorescence, Western Blot, Expressing, Immunoprecipitation, Staining, Ligation, Negative Control

Journal: iScience

Article Title: Aβ∗56 is a stable oligomer that impairs memory function in mice

doi: 10.1016/j.isci.2024.109239

Figure Lengend Snippet: Age-related presence of ∼56-kDa, SDS-stable, water-soluble, Aβ-containing entities in Tg2576 mice (A) A schematic illustration of epitopes of capturing and detecting antibodies used in immunoprecipitation (IP)/western blotting (WB) for (B). Capturing antibody: rabbit monoclonal D8Q7I that is directed against the C-terminus of Aβ(x-40), detecting antibody: biotinylated mouse monoclonal 6E10 that is directed against Aβ(3-8). APP, amyloid precursor protein; Aβ, amyloid-β. (B) A representative IP/WB analysis showing that D8Q7I-precipitated entities electrophoresed at ∼56 kDa (arrow) are detected by biotinylated 6E10 (6E10-biotin) in 10-month-old and 17-month-old Tg2576 (+) mice but not in 4- to 5-month-old Tg2576 (+) mice, in 17-month-old littermates of Tg2576 mice (−), or when no Tg2576 brain extracts were used (Ab only). No ∼56-kDa entities are detected when generic rabbit immunoglobulin G (RbIgG) was used to precipitate brain extracts (+°, combined brain extracts of the four 10-month- and 17-month-old Tg2576 mice, each contributing 25% of the total proteins). Ab, capturing antibody D8Q7I. Syn Aβ 40 , synthetic Aβ(1-40) (4 ng), serving as a positive control for WB. #, monomeric Aβ(x-40). ∗, non-specifically detected entities. kDa, kilo-Daltons. Std., protein standards; and mo., months. (C) The percentage of Tg2576 mice expressing ∼56-kDa entities increases with age. Non-transgenic littermates (−) of Tg2576 and rTg9191 mice, 2- to –5-month-old Tg2576 mice (+), and 24-month-old rTg9191 mice (+) do not express the ∼56-kDa entity. The ages (mo., months) shown in the figure are defined as follows: 2–16 months, 1.5–16.4 months; 2–5 months, 1.5–5.4 months; 6–11 months, 5.5–11.4 months; 12–17 months, 11.5–17.4 months; 20–25 months, 19.5–25.4 months; and 24 months, 23.5–24.4 months. The total number ( N ) of mice of each age group is listed. Detailed information for the calculation is listed in Table S1 . See also Figures S1 , , , and ; Tables S1 , , and .

Article Snippet: Rabbit polyclonal biotinylated anti-amyloid oligomers , StressMarq Biosciences , Cat#SPC-506D-BI; Clone#A11; RRID: AB_10962958.

Techniques: Immunoprecipitation, Western Blot, Positive Control, Expressing, Transgenic Assay

Journal: iScience

Article Title: Aβ∗56 is a stable oligomer that impairs memory function in mice

doi: 10.1016/j.isci.2024.109239

Figure Lengend Snippet: Canonical Aβ(1-40) is present in the ∼56-kDa, SDS-stable, water-soluble, Aβ-containing entities in Tg2576 mice (A) A schematic illustration of epitopes of capturing and detecting antibodies used in immunoprecipitation (IP)/western blotting (WB) for (B). Capturing antibody: rabbit monoclonal D8Q7I; detecting antibody: biotinylated mouse monoclonal 82E1 that is directed against the N-terminus of Aβ(1-x). APP, amyloid precursor protein; Aβ, amyloid-β. (B) A representative IP/WB analysis showing that D8Q7I-precipitated entities electrophoresed at ∼56 kDa (arrow) are detected by biotinylated 82E1 (82E1-biotin) in 8- to 12-month-old Tg2576 mice (+) but not in age-matched, non-transgenic littermates (−). No ∼56-kDa, D8Q7I-precipitated entities are detected when no Tg2576 brain extracts were used (Ab only). No ∼56-kDa entities are detected in Tg2576 mice when generic rabbit immunoglobulin G (RbIgG) was used to precipitate brain extracts (+°, combined brain extracts of the seven Tg2576 mice, each contributing 14.3% of the total proteins). Ab, capturing antibody D8Q7I. Syn AβpE3-42, synthetic Aβ 42 with the 3 rd amino acid being a pyroglutamate (30 ng), serving as a negative control for WB, and Syn Aβ 40/42 , synthetic Aβ(1-40) (5 ng) and Aβ(1-42) (5 ng), serving as a positive control for WB. #, monomeric Aβ; $, dimeric Syn Aβ 40/42 ; @, trimeric Syn Aβ 40/42 ; and ∗, non-specifically detected entities. kDa, kilo-Daltons. See also Figures S31–S33 and ; Tables S1 , , and .

Article Snippet: Rabbit polyclonal biotinylated anti-amyloid oligomers , StressMarq Biosciences , Cat#SPC-506D-BI; Clone#A11; RRID: AB_10962958.

Techniques: Immunoprecipitation, Western Blot, Transgenic Assay, Negative Control, Positive Control

Journal: iScience

Article Title: Aβ∗56 is a stable oligomer that impairs memory function in mice

doi: 10.1016/j.isci.2024.109239

Figure Lengend Snippet: The ∼56-kDa, Aβ(1-40)-containing entities are not artificially formed by exposure to SDS (A) Representative western blotting (WB) analyses showing the detection of the ∼56-kDa, Aβ-containing entities (arrow, fractions [Frac.] 47–67), ∼14-kDa entities (%, Frac. 69–75), and monomeric Aβ (#, frac. 37–55 and 75–81) in odd-numbered size-exclusion chromatography (SEC) fractions (Frac. 33–95) of 10- to 11-month-old Tg2576 brain extracts using biotinylated 82E1 (82E1-biotin). The four WB images (i.e., Frac. 33–47, 49–63, 65–79, and 81–95) shown were obtained using the same exposure time (20 s), and the WB band patterns and intensities of the input material (2.5% (w/v) of the brain extracts used for SEC) within each of the four WB images are comparable. A WB of the input is shown in the far-left panel. To better visualize monomeric Aβ, WB images at a longer exposure time (100 s) are shown in lower panels. The elution profile of biomolecule standards (Std.) of varied sizes is shown above the WB images. kDa, kilo-Daltons. (B) Quantitative analyses of levels of the ∼56-kDa, 82E1-reactive entities and monomeric Aβ in SEC fractions. The level of the ∼56-kDa entities in each fraction is normalized to its highest level in fraction 63 and of monomeric Aβ (Aβ mono.) in each fraction is normalized to its highest level in fraction 77. (C) Representative WB analyses showing the detection of the ∼56-kDa, Aβ-containing entities (arrows, Frac. 52–72), ∼14-kDa entities (%, Frac. 68–74), and monomeric Aβ (#, Frac. 36–48 and 76–78) in even-numbered SEC fractions (Frac. 34–96) of 10- to 11-month-old Tg2576 brain extracts using biotinylated D8Q7I (D8Q7I-biotin). The four WB images (i.e., Frac. 34–48, 50–64, 66–80, and 82–96) were obtained using the same exposure time (50 s), and the WB band patterns and intensities of the input material (2.5% [w/v] of the brain extracts used for SEC) within each of the four WB images are comparable. A WB of the input is shown in the far-left panel. To better visualize monomeric Aβ, WB images at a longer exposure time (100 s) are shown in lower panels. The elution profile of biomolecule standards (Std.) of varied sizes is shown above the WB images. kDa, kilo-Daltons. (D) Quantitative analyses of levels of the ∼56-kDa, D8Q7I-reactive entities and monomeric Aβ in SEC fractions. Levels of the ∼56-kDa entities in each fraction are normalized to their highest level in fraction 56 (lower band) and fraction 64 (upper band), and the level of monomeric Aβ (Aβ mono.) in each fraction is normalized to its highest level in fraction 38. See also Figures S35–S37 ; Tables S1 , , and .

Article Snippet: Rabbit polyclonal biotinylated anti-amyloid oligomers , StressMarq Biosciences , Cat#SPC-506D-BI; Clone#A11; RRID: AB_10962958.

Techniques: Western Blot, Size-exclusion Chromatography

Journal: iScience

Article Title: Aβ∗56 is a stable oligomer that impairs memory function in mice

doi: 10.1016/j.isci.2024.109239

Figure Lengend Snippet: Immunopurified Aβ∗56 is SDS-stable, contains canonical Aβ(1-40), and is recognized by A11, anti-oligomer antibodies (A) A representative western blotting (WB) analysis showing under sodium dodecyl sulfate (SDS) denaturing conditions the detection of the ∼56-kDa, D8Q7I-purified, 82E1-reactive entities (arrow), monomeric Aβ (#), and multiple entities of intermediate sizes (10–50 kDa, vertical line) in the brains of 15- to 22-month-old Tg2576 mice (+) but not age-matched littermates (−). (B) A representative WB analysis showing that under SDS denaturing conditions, the ∼56-kDa, D8Q7I-purified entities (arrow), but no other entities, bind to the biotinylated anti-oligomer antibody A11 (A11-biotin) in the brains of 15- to 21-month-old Tg2576 mice (+) but not age-matched littermates (−). (C) A representative WB analysis showing that under SDS semi-denaturing conditions, the D8Q7I-purified, ∼56-kDa (arrows), ∼27-kDa (<), ∼22-kDa ($), and ∼10-15-kDa (vertical line) entities are reactive to biotinylated anti-oligomer antibody A11 (A11-biotin) in the brains of 15- to 22-month-old Tg2576 mice (+) but not age-matched littermates (−). kDa, kilo-Daltons. ∗non-specifically detected entities. See also Figures S38 and ; Tables S1 , , and .

Article Snippet: Rabbit polyclonal biotinylated anti-amyloid oligomers , StressMarq Biosciences , Cat#SPC-506D-BI; Clone#A11; RRID: AB_10962958.

Techniques: Western Blot, Purification

Journal: iScience

Article Title: Aβ∗56 is a stable oligomer that impairs memory function in mice

doi: 10.1016/j.isci.2024.109239

Figure Lengend Snippet: Aβ∗56 isolated from Tg2576 mice impairs memory function (A) A representative western blotting (WB) analysis showing the detection of Aβ∗56 isolated from brains of 17- to 22-month-old Tg2576 mice following immunoaffinity purification coupled to SDS-PAGE/electro-elution. The quantity and purity of isolated Aβ∗56 (arrow, 2 μL loaded) used for surgical injection and mouse memory tests (D and E) were assessed by WB with various amounts of synthetic monomeric Aβ(1-40) (Syn Aβ 40 ) serving as quantification standards. Aβ∗56, but no other Aβ species, is detected by biotinylated 82E1 (82E1-biotin). #, monomeric Aβ(1-40); and $, dimeric Aβ(1-40). (B) Densitometry-based quantification of Aβ∗56. The linear relationship between the amounts of 82E1-reactive proteins and band intensities in (A) was established using synthetic Aβ(1-40) standards. By fitting the band intensity of Aβ∗56 to the standard curve, the concentration of Aβ∗56 was determined to be 3.9 ng/μL (i.e., 70 nM, assuming that the stoichiometry of 82E1 binding to monomeric Aβ(1-40) is equal to that of 82E1 binding to Aβ∗56). (C) A representative silver stain analysis assessing the purity of isolated Aβ∗56 (arrow, 5 μL loaded) used for the study of mouse memory (D and E). (D) A schematic illustration of bilateral intra-hippocampal injections of Aβ∗56. (E) Healthy 4- to 7-month-old wild-type mice injected with Aβ∗56 exhibit lower ( U = 19, p = 0.0159; two-tailed Mann-Whitney U test) discrimination indices compared with those injected with vehicle (phosphate-buffered saline) in an object recognition test. The scatter plot with bar graph represents data of individual mice plus group median ± interquartile range. The numbers of mice used for the vehicle and Aβ∗56 groups are shown in parentheses. Veh, vehicle; kDa, kilo-Dalton.

Article Snippet: Rabbit polyclonal biotinylated anti-amyloid oligomers , StressMarq Biosciences , Cat#SPC-506D-BI; Clone#A11; RRID: AB_10962958.

Techniques: Isolation, Western Blot, Immunoaffinity Purification, SDS Page, Injection, Concentration Assay, Binding Assay, Silver Staining, Two Tailed Test, MANN-WHITNEY, Saline

Journal: iScience

Article Title: Aβ∗56 is a stable oligomer that impairs memory function in mice

doi: 10.1016/j.isci.2024.109239

Figure Lengend Snippet: Aβ∗56 is present in two other mouse models of AD (A) A schematic illustration of epitopes of capturing and detecting antibodies used in immunoprecipitation (IP)/western blotting (WB) for (B–E). Capturing antibody: rabbit monoclonal D8Q7I that is directed against the C-terminus of Aβ(x-40) or rabbit monoclonal D3E10 that is directed against the C-terminus of Aβ(x-42), detecting antibody: biotinylated mouse monoclonal 82E1 that is directed against the N-terminus of Aβ(1-x). APP, amyloid precursor protein; Aβ, amyloid-β. (B) Representative IP/WB analyses showing that D8Q7I-precipitated entities electrophoresed at ∼56 kDa (arrow) are detected by biotinylated 82E1 (82E1-biotin) in 17-month-old Tg2576 (+’) mice (serving as a positive control for IP/WB) but not in 9-month-old 5xFAD (+) mice, in age-matched non-transgenic littermates (−) of 5xFAD mice, or when no brain extracts were used (Ab only). No ∼56-kDa entities are detected when generic rabbit immunoglobulin G (RbIgG) was used to precipitate brain extracts (+°, combined brain extracts of the four 5xFAD mice, each contributing 25% of the total proteins). Ab, capturing antibody D8Q7I. Syn Aβ 40 , synthetic Aβ(1-40) (1 ng), serving as a positive control for WB. #, monomeric Aβ(x-40). (C) Representative IP/WB analyses showing that D3E10-precipitated entities electrophoresed at ∼56 kDa (arrow) are detected by 82E1-biotin in 9-month-old 5xFAD (+) mice but not in age-matched non-transgenic littermates (−) of 5xFAD mice or when no brain extracts were used (Ab only). No ∼56-kDa entities are detected when RbIgG was used to precipitate brain extracts (+°, combined brain extracts of the four 5xFAD mice, each contributing 25% of the total proteins). Ab, capturing antibody D3E10. Syn Aβ 42 , synthetic Aβ(1-42) (5 ng), serving as a positive control for WB. #, monomeric Aβ(x-42); $, dimeric Syn Aβ 42 ; @, trimeric Syn Aβ 42 . (D) Representative IP/WB analyses showing that D8Q7I-precipitated entities electrophoresed at ∼56 kDa (arrow) are detected by 82E1-biotin in 17-month-oldTg2576 (+’, serving as a positive control for IP/WB) and 10- to 11-month-old APP/TTA (+) mice but not in age-matched non-transgenic littermates (−) of APP/TTA mice or when no brain extracts were used (Ab only). No ∼56-kDa entities are detected when RbIgG was used to precipitate brain extracts (+°, combined brain extracts of the four APP/TTA mice, each contributing 25% of the total proteins). Ab, capturing antibody D8Q7I. Syn Aβ 40 , synthetic Aβ(1-40) (1 ng), serving as a positive control for WB. #, monomeric Aβ(x-40). (E) Representative IP/WB analyses showing that D3E10-precipitated entities electrophoresed at ∼56 kDa (arrow) are detected by 82E1-biotin in 10- to 11-month-old APP/TTA (+) mice but not in age-matched non-transgenic littermates (−) of APP/TTA mice or when no brain extracts were used (Ab only). No ∼56-kDa entities are detected when RbIgG was used to precipitate brain extracts (+°, combined brain extracts of the four APP/TTA mice, each contributing 25% of the total proteins). Ab, capturing antibody D3E10. Syn Aβ 42 , synthetic Aβ(1-42) (5 ng), serving as a positive control for WB. #, monomeric Aβ(x-42); $, dimeric Syn Aβ 42 ; @, trimeric Syn Aβ 42 . For (B–E), ∗, non-specifically detected entities and kDa, kilo-Daltons.

Article Snippet: Rabbit polyclonal biotinylated anti-amyloid oligomers , StressMarq Biosciences , Cat#SPC-506D-BI; Clone#A11; RRID: AB_10962958.

Techniques: Immunoprecipitation, Western Blot, Positive Control, Transgenic Assay

Journal: iScience

Article Title: Aβ∗56 is a stable oligomer that impairs memory function in mice

doi: 10.1016/j.isci.2024.109239

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal biotinylated anti-amyloid oligomers , StressMarq Biosciences , Cat#SPC-506D-BI; Clone#A11; RRID: AB_10962958.

Techniques: Recombinant, Software, Chromatography, Imaging