Journal: bioRxiv

Article Title: Arginyltransferase1 drives a mitochondria-dependent program to induce cell death

doi: 10.1101/2024.11.22.624728

Figure Lengend Snippet: (A) Representative Western blot showing total levels of Ate1-GFP in W303 wild-type strain treated either with or without 5%NaN 3 for 30 minutes. Ate1-GFP expression was induced by the addition of 0.5% galactose and incubation for 3 hours at 30°C. Empty vector serves as a negative control. Alpha tubulin is used for protein loading control. (B) Left panel; representative Western blot showing Ate1-GFP levels in purified mitochondrial fractions, which was treated with proteinase K (5 μg/ml) to remove proteins that are not protected by the mitochondrial membrane. Alpha-tubulin is used as a marker for cytosolic protein contamination, while mitochondrial intermembrane space protein Cmc2 serves as a marker for mitochondrial proteins. Right panel: showing the quantification of mitochondrial Ate1-GFP levels normalized to mitochondrial protein Cmc2. Error bar denotes SD (N=4), p values were calculated by two tailed student t-test.

Article Snippet: Western blot blocking reagent was obtained from Roche (catalogue number 75255200) The primary antibodies include: monoclonal mouse anti-GFP (from Roche, clone 7.1 and 13.1, Cat# 11814460001) Rabbit anti-yeast alpha tubulin (Abcam EPR13799) anti-yeast-phosphoglycerate kinase1 (Pgk1) (Thermofischer scientific # 459250), Rabbit anti-yeast Cmc2 (gift from Dr. Antonio Barrientos) mouse anti-Grp78 (SCBT# HDEL Antibody (2E7): sc-53472) HSP70 Monoclonal antibody (Proteintech catalogue# 66183-1) The custom-produced rabbit anti-RDD antibody was ordered from Genscript INC as described in our previous work [ ].

Techniques: Western Blot, Expressing, Incubation, Plasmid Preparation, Negative Control, Control, Purification, Membrane, Marker, Two Tailed Test

Journal: bioRxiv

Article Title: Arginyltransferase1 drives a mitochondria-dependent program to induce cell death

doi: 10.1101/2024.11.22.624728

Figure Lengend Snippet: (A) Yeast cells containing Ate1-GFP and Ate1-GFP-Ras2 that are driven by galactose-inducible promoters (pGal) were allowed to express for 6 hours and then briefly treated with oxidative stressor H 2 O 2 for 10 minutes before the microscopic images were taken for the green fluorescence channel. The regular Ate1-GFP forms puncta-like structures similar as shown in , while Ate1-GFP-Ras2 remains localized to the periphery of the cells. (B) Scheme illustrating the principle of the reporter for the arginylation activity inside yeast cells. The N-terminal ubiquitin domain of the reporter protein DD-β15-mCherryFP will be promptly removed by endogenous de-ubiquitination (de-Ub) enzymes in the cell, exposing the penultimate peptide DD-β15, which is derived from the N-terminus of mouse β-actin and is known to be arginylated in vivo [ , ]. The arginylated N-terminus can be recognized with a specific antibody anti-RDD. Antibodies for mCherryFP (mChFP) and GFP can be used to probe the levels of the reporter protein and the GFP-fused ATE1, respectively. (C) To test the arginylation activity of different forms of Ate1 (Ate1-GFP or Ate1-GFP-Ras2), they were expressed in ate1 Δ yeast (to avoid the interference of endogenous ATE1), which was also simultaneously expressing the reporter protein DD-β15-mCherryFP. The arginylation level of the reporter protein was measured as described in (B). Pgk1 serves as loading controls for the yeast proteins. (D) Growth test of ate1 Δ yeast cells carrying either the empty expression vector or pGAL1:Ate1-GFP and pGAL1:Ate1-GFP-Ras2 was conducted by a serial dilution growth assay on either plate containing glucose or galactose, where the expression of Ate1 is not induced or induced, respectively. (E) Representative Western blots showing the expression levels and distributions of different Ate1 constructs (Ate1-GFP and the mitochondrial matrix-targeting Mt-Ate1-GFP) in total cell lysate (left panel) and in purified mitochondrial fractions (right panel). Vector alone served as a negative control. The expression of both the Ate1 construts was achieved by adding 0.5% galactose and incubated for 3hrs at 30 o C. The expressions and subcellular distributions Ate1-GFP and Mt-Ate1-GFP were probed by anti-GFP. The level of Pgk11 and Cmc2 were used as loading controls for total proteins or mitochondrial proteins, respectively. (F) Growth test of ate1 Δ yeast cells carrying either the empty expression vector, pGAL1:Ate1-GFP, or the mitochondria matrix localized Ate1 (pGAL1: Mt-Ate1-GFP) by a serial dilution growth assay on either plate containing galactose or glucose for the induced expression (or not) of Ate1. Note that the concentration of galactose (0.5%) is lower than elsewhere to allow the display of the difference between the Ate1-GFP and mt-Ate1-GFP. Plates were incubated at 30°C and images were taken after 3 days.

Article Snippet: Western blot blocking reagent was obtained from Roche (catalogue number 75255200) The primary antibodies include: monoclonal mouse anti-GFP (from Roche, clone 7.1 and 13.1, Cat# 11814460001) Rabbit anti-yeast alpha tubulin (Abcam EPR13799) anti-yeast-phosphoglycerate kinase1 (Pgk1) (Thermofischer scientific # 459250), Rabbit anti-yeast Cmc2 (gift from Dr. Antonio Barrientos) mouse anti-Grp78 (SCBT# HDEL Antibody (2E7): sc-53472) HSP70 Monoclonal antibody (Proteintech catalogue# 66183-1) The custom-produced rabbit anti-RDD antibody was ordered from Genscript INC as described in our previous work [ ].

Techniques: Fluorescence, Activity Assay, Derivative Assay, In Vivo, Expressing, Plasmid Preparation, Serial Dilution, Growth Assay, Western Blot, Construct, Purification, Negative Control, Incubation, Concentration Assay

Journal: bioRxiv

Article Title: Arginyltransferase1 drives a mitochondria-dependent program to induce cell death

doi: 10.1101/2024.11.22.624728

Figure Lengend Snippet: A) Top panel shows the representative Western blot for the level of Grp78/HDEL, a maker of the endoplasmic reticulum stress response, in yeast cells carrying the pYES2-pGAL: ATE1-6xHis- URA3 expression vector or the empty control vector, which were induced with 2% galactose for 6h in liquid media. Pgk1 is used as loading controls. Ate1 was probed with anti-GFP. Bottom panel is a graph indicating the fold differences in GRP78 levels between the Ate1 overexpression (OE) and vector control. Error bar denotes SEM (N=3). B) Top panel shows representative Western blot for the level of full-length Atg8-GFP and the derivative GFP, which is resulted form proteolysis in autophagy. The yeast cells carrying the pYES2-pGAL: ATE1-6xHis- URA3 expression vector or the empty control vector were induced with 2% galactose for 6h in liquid media. Pgk1 is a loading control. Bottom panel is a graph indicating the fold differences in the ratio between GFP and the full-length Atg8-GFP, which reflects the activity of autophagy, in the presence of Ate1 overexpression (OE) and vector control. Error bar denotes SEM (N=3).

Article Snippet: Western blot blocking reagent was obtained from Roche (catalogue number 75255200) The primary antibodies include: monoclonal mouse anti-GFP (from Roche, clone 7.1 and 13.1, Cat# 11814460001) Rabbit anti-yeast alpha tubulin (Abcam EPR13799) anti-yeast-phosphoglycerate kinase1 (Pgk1) (Thermofischer scientific # 459250), Rabbit anti-yeast Cmc2 (gift from Dr. Antonio Barrientos) mouse anti-Grp78 (SCBT# HDEL Antibody (2E7): sc-53472) HSP70 Monoclonal antibody (Proteintech catalogue# 66183-1) The custom-produced rabbit anti-RDD antibody was ordered from Genscript INC as described in our previous work [ ].

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Over Expression, Activity Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: In Search of the Identity of the XAP-1 Antigen: A Protein Localized to Cone Outer Segments

doi: 10.1167/iovs.09-4286

Figure Lengend Snippet: LC-MS/MS results obtained from the protein band corresponding to the XAP-1 antigen. (A) The MS/MS spectrum of a peptide from the LC-MS/MS analysis. It contains product ions of the b- and y- series that matched peptide sequence ITPSYVAFTPEGER from Grp78 from Mus musculus. (B) The m/z ratios for theoretical product ions of the y- and b- series. Product ions that were observed in the MS/MS spectrum are shown in bold.

Article Snippet: Blot A was incubated in rabbit anti-human Grp78 (1:1000 dilution; Anaspec) overnight at 4°C.

Techniques: Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Sequencing

Journal: Investigative Ophthalmology & Visual Science

Article Title: In Search of the Identity of the XAP-1 Antigen: A Protein Localized to Cone Outer Segments

doi: 10.1167/iovs.09-4286

Figure Lengend Snippet: Protein sequence of Mus musculus Grp78. The peptide from our MS/MS analysis is shown in gray text and represents 2.14% coverage.

Article Snippet: Blot A was incubated in rabbit anti-human Grp78 (1:1000 dilution; Anaspec) overnight at 4°C.

Techniques: Sequencing, Tandem Mass Spectroscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: In Search of the Identity of the XAP-1 Antigen: A Protein Localized to Cone Outer Segments

doi: 10.1167/iovs.09-4286

Figure Lengend Snippet: Depletion of IgM and Grp78 from the outer segment–enriched preparation. IgM and Grp78 were depleted from outer segment (OS) cell extracts. IgM and the XAP-1 antigen are shown in red, whereas Grp78 is shown in green. Overlap of the signals (E, F, yellow). A small amount of IgM was detected after one round of depletion, but none remained after a second round (A). Both the XAP-1 antigen (B) and Grp78 (C, D) remained in the sample after all IgM was removed. The XAP-1 antigen and Grp78 overlap at an identical relative molecular mass (F). Depletion of Grp78 (C, D) abolished the signal obtained with the XAP-1 antibody (B).

Article Snippet: Blot A was incubated in rabbit anti-human Grp78 (1:1000 dilution; Anaspec) overnight at 4°C.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: In Search of the Identity of the XAP-1 Antigen: A Protein Localized to Cone Outer Segments

doi: 10.1167/iovs.09-4286

Figure Lengend Snippet: Immunohistochemical localization of Grp78 in the retinas of mouse and frog. After antigen retrieval, retinas from C57BL6/J mice were immunostained using anti-Grp78 (A, green), PNA (B, red), and iodide (C, blue). (C) Composite image. Yellow (arrows) indicates areas of overlap of the Grp78 and PNA, indicating that in the outer retina, Grp78 is localized to cone photoreceptors in a pattern identical with that of the XAP-1 antigen. (C, inset) Higher magnification image of the area within the inset in C. In the mouse, Grp78 is also found in all cell layers of the retina in a location corresponding to ER. (D) Examination of the frog retina revealed a different pattern. The peripheries of both rod and cone photoreceptor outer segments and the inner segments are immunolabeled in this species. Scale bar, 10 μm. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Article Snippet: Blot A was incubated in rabbit anti-human Grp78 (1:1000 dilution; Anaspec) overnight at 4°C.

Techniques: Immunohistochemical staining, Immunolabeling

Journal: Biocell

Article Title: Stretch Enhances Secretion of Extracellular Vehicles from Airway Smooth Muscle Cells via Endoplasmic Reticulum Stress Signaling in Relation to Ventilator-Induced Lung Injury

doi: 10.32604/biocell.2025.063869

Figure Lengend Snippet: Figure 2: The effect of stretch on EVs and endoplasmic reticulum (ER) stress in ASMCs. (A) Left to right: representative immunofluorescence images of CD63/HSPA5 in ASMCs acquired with confocal microscopy (×100 objective), the quantified CD63/HSPA5 puncta number inside ASMCs (ImageJ), and Pearson coefficient of the pixel-intensity correlation of CD63/HSPA5 in ASMCs (ImageJ, scale bar = 200 μm); (B) Protein expression of CD63/HSPA5 in ASMCs (WB); (C) Size distribution and quantified counts, as well as protein concentration of EVs in ASMCs (NTA and BCA). ER: endoplasmic reticulum; HSPA5: biomarker protein of ER stress; TUDCA: tauroursodeoxycholic acid, ER stress inhibitor; NTA, nanoparticle tracking analysis; BCA: bicinchoninic acid assay; data present as means ± SD, experiments were repeated three times (n = 3), **p < 0.01 compared with Static, ##p < 0.01 compared with Stretch

Article Snippet: Afterwards it was immersed in 10% horse serum (Sigma, #H0146) in PBS as a blocking solution for 1 h at RT, washed 3 times in PBS for 5 min, and then incubated with HSPA5 antibody (1:100, #BA2042, BOSTER) in PBS containing 5% horse serum for 1 h at RT.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing, Protein Concentration, Biomarker Discovery, Acid Assay

Journal: Biocell

Article Title: Stretch Enhances Secretion of Extracellular Vehicles from Airway Smooth Muscle Cells via Endoplasmic Reticulum Stress Signaling in Relation to Ventilator-Induced Lung Injury

doi: 10.32604/biocell.2025.063869

Figure Lengend Snippet: Figure 6: The effect of mechanical ventilation on secretion of EVs, ER stress, airway inflammation and injury in mouse models of VILI. (A) Representative images of lung tissue slides with hematoxylin and eosin (HE) or immunohisto- chemistry (IHC, HSPA5) staining (scale bar = 100 μm, arrows indicate alveolar collapse, arrowhead indicate HSPA5 expression). Healthy, MV, MV + TUDCA/GW4869 indicates mice breathed spontaneously, under 18 mL/kg mechanical ventilation (MV), MV with pretreatment of TUDCA/GW4869, respectively; (B) Protein expression of HSPA5 in lung tissue with IHC staining from C57BL/6 mice treated with different conditions, respectively; (C–E) Size distribution and quantified count, HSPA5, as well as protein concentration of EVs isolated from bronchoalveolar lavage fluid (BALF) of mice treated with different conditions; (F) Relative TGF-β1 and IL-10 secretion in BALF from mice treated with different conditions. Data present as means ± SD, experiments were repeated six times (n = 6), ** p < 0.01 compared with Healthy, #p < 0.05, ##p < 0.01 compared with MV

Article Snippet: Afterwards it was immersed in 10% horse serum (Sigma, #H0146) in PBS as a blocking solution for 1 h at RT, washed 3 times in PBS for 5 min, and then incubated with HSPA5 antibody (1:100, #BA2042, BOSTER) in PBS containing 5% horse serum for 1 h at RT.

Techniques: Immunohistochemistry, Staining, Expressing, Protein Concentration, Isolation

Journal: Pharmaceutics

Article Title: The Anticancer Ruthenium Compound BOLD-100 Targets Glycolysis and Generates a Metabolic Vulnerability towards Glucose Deprivation

doi: 10.3390/pharmaceutics14020238

Figure Lengend Snippet: Glucose deprivation by 2-DG differentially regulates ER stress and leads to apoptotic cell death upon combination with BOLD-100. ( a ) Representative composite images show morphological changes of HCT116 or HCTR cells detected with dual staining of Hoechst 33342/PI. Cells were treated for 72 h with DMSO (control, equivalent to BOLD-100), 2.5 mM 2-DG, 100 µM BOLD-100 or their combination and imaged by fluorescence microscopy (magnification 20×). Grey arrows indicate examples of mitotic nuclei. Green arrows indicate live cells with apoptotic nuclei. Red arrows indicate dead cells with late apoptotic nuclei. ( b ) Quantification of treatment-respective early and late apoptotic nuclei depicted in ( a ). Statistical significance of differences was calculated using a two-way ANOVA with Tukey´s multiple comparisons test: * p < 0.05, **** p < 0.0001; ns: non-significant. ( c ) Expression levels of GRP78, peIF2A (Ser51), eIF2A, HSP70, and PARP after 24 h of treatment of HCT116 and HCTR cells with the indicated concentrations of BOLD-100, 2-DG, or their combination, analyzed by Western blotting. Two different control states were included, i.e., medium control without DMSO for 2-DG and with DMSO as a solvent control for BOLD-100. β-actin served as the loading control. The numbers below indicate quantified Western blot signal intensities normalized to their respective controls: blue, medium; black, DMSO; and red, HCTR vs. HCT116 cells.

Article Snippet: The primary antibodies Anti-GRP78 (C50B12) (#3177, dilution 1:1000), p-eIF2A (Ser51) (#9721S, 1:1000), eIF2A (L57A5) (#2103, 1:1000), HSP70 (#4872, 1:1000), PARP (46D11) (#9532, 1:1000), beclin-1 (#3495, 1:1000), p62/SQSTM1 (#8025, 1:500), and LC3 B I/II (#12741, 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Control, Fluorescence, Microscopy, Expressing, Western Blot, Solvent