Journal: Life Science Alliance

Article Title: Glycine cleavage system determines the fate of pluripotent stem cells via the regulation of senescence and epigenetic modifications

doi: 10.26508/lsa.201900413

Figure Lengend Snippet: (A) Western blot analysis of the expression of senescence-related genes in V6.5 cells treated with various concentration gradients of MG. ACTIN served as the loading control. (B) Western blot analysis of the expression of senescence-related genes in reprogramming cells treated with MG. ACTIN served as the loading control. (C) Reprogramming cells were treated with MG or control, and the SA-β-gal-positive cells were counted. The data were presented as the mean ± SD. * P < 0.05 compared with CTR; t test. Scale bars, 100 μm. (D) MG was added to the medium of MEF cells starting from 2 d after infection with virus expressing the four factors. AP staining (upper panel) showed the iPSC colonies formed. The AP- and Ssea1-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. * P < 0.05 compared with CTR; t test. (E) Western blot analysis of the expression of senescence-related genes and argpyrimidine in V6.5 cells stably expressing shGldc or the NTC. ACTIN served as the loading control. (F) Western blot analysis of the expression of senescence-related genes and argpyrimidine in reprogramming cells expressing shGldc the NTC at various time points. ACTIN served as the loading control. (G) Reprogramming cells were infected with viruses expressing shGldc or the NTC, and SA-β-gal–positive cells were counted. The data were presented as the mean ± SD. * P < 0.05 compared with the indicated group; t test. Scale bars, 100 μm. (H) V6.5 cells stably expressing shGldc or the NTC were further infected with viruses expressing pSIN-3×flag-Glo1 or were supplemented with 1 mM carnosine, followed by qRT-PCR analysis of senescence-related genes. The data were presented as the mean ± SD of three independent experiments. * P < 0.05 compared with the NTC, # P < 0.05 compared with the Gldc knockdown group; t test. (I) V6.5 cells stably expressing shGldc or the NTC were further infected with viruses expressing pSIN-3×flag-Glo1 or EV, followed by Western blot analysis of senescence-related genes and argpyrimidine modification. ACTIN served as the loading control. (J) Reprogramming cells expressing shGldc or the NTC were further infected with viruses expressing pSIN-3×flag-Glo1 or EV, followed by Western blot analysis of senescence-related genes and argpyrimidine modification. ACTIN served as the loading control. (K) Reprogramming cells expressing shGldc or the NTC were further infected with viruses expressing pSIN-3×flag-Glo1 or EV, and SA-β-gal–positive cells were counted. The data were presented as the mean ± SD. * P < 0.05 compared with the NTC, # P < 0.05 compared with the Gldc knockdown group; t test. Scale bars, 100 μm. (L) Reprogramming cells expressing shGldc or the NTC were further infected with viruses expressing pSIN-3×flag-Glo1. AP staining (upper panel) showed the iPSC colonies formed. The AP- and Ssea1-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. * P < 0.05 compared with the NTC, # P < 0.05 compared with the Gldc knockdown group; t test. (M) MEFs were induced with the indicated factors. AP staining (upper panel) showed the iPSC colonies formed. The AP- and Ssea1-positive iPSC colonies were counted (lower panel). The data were presented as the mean ± SD. * P < 0.05 compared with the NTC, # P < 0.05 compared with the Gldc knockdown group; t test. NS, not significant. Source data are available for this figure.

Article Snippet: Primary antibodies against the following proteins were used: OCT4 (from Stemgent); SOX2 (from Millipore); MYC (from Epitomics); KLF4, SSEA1, SHMT1, SHMT2, TDH, AMT, H3, P15, and MG (from Abcam); H3K4me3, H3K9me3, H3K27me3, and H3K36me3 (from Cell Signaling Technology); FLAG (from Sigma-Aldrich); ACTIN, phosphoglycerate dehydrogenase, PSAT1, PSPH, GCAT, GLDC, GCSH, P16, P21, and GLO1 (from Proteintech).

Techniques: Western Blot, Expressing, Concentration Assay, Control, Infection, Virus, Staining, Stable Transfection, Quantitative RT-PCR, Knockdown, Modification

Journal: Advanced Science

Article Title: Targeting Methylglyoxal Metabolism to Enhance Ferroptosis Sensitivity in Tumor Therapy

doi: 10.1002/advs.202505356

Figure Lengend Snippet: Proteasomal degradation of GLO1 mediated by PJA1 promotes ferroptosis. a,b) Analysis of GLO1 protein and mRNA expression in the indicated PANC1 cells treated with erastin (5 µ m ) or RSL3 (500 n m ) for 6–24 h. c) Western blot analysis of protein expression in PANC1 and HT1080 cells after treatment with erastin (5 µ m ) or RSL3 (500 n m ) for 24 h, with or without MG132 (5 µ m ) or HCQ (10 µ m ). d) Bioinformatic analysis using the STRING database identified PJA1 and CUL7 as potential E3 ligases interacting with GLO1. These interactions were experimentally validated in PANC1 cells by immunoprecipitation (IP) assays. e) Western blot analysis of GLO1 protein expression in the indicated PANC1 and HT1080 cells treated with erastin (5 µ m ) or RSL3 (500 n m ) for 24 h. f–h) Analysis of cell viability, intracellular MGO and MDA concentrations in the indicated PANC1 and HT1080 cells treated with erastin (5 µ m ) or RSL3 (500 n m ) for 6–24 h. i) Indicated PANC1 cells were treated with erastin (5 µM) and cycloheximide (CHX, 100 µg mL −1 ) for the indicated times (0–12 h). Whole‐cell lysates were subjected to immunoblotting for GLO1 and ACTB (loading control). j–l) The effects of PARK7 knockout on erastin (5 µ m )‐induced growth inhibition and MGO accumulation after 24 h in PANC1 cells. All semi‐quantitative data are presented as means ± SD for n = 3 biologically independent samples (b,f,g,h,k,l). Statistical analyses were conducted using ANOVA with Tukey's multiple comparisons test (f–h).

Article Snippet: Plasmids containing the wild‐type PJA1 (OriGene, RC224490) and GLO1 (OriGene, RC203826) coding sequences served as templates for the mutagenesis reactions.

Techniques: Expressing, Western Blot, Immunoprecipitation, Control, Knock-Out, Inhibition

Journal: Advanced Science

Article Title: Targeting Methylglyoxal Metabolism to Enhance Ferroptosis Sensitivity in Tumor Therapy

doi: 10.1002/advs.202505356

Figure Lengend Snippet: Cysteine 598 of PJA1 is required for the degradation of GLO1 and subsequent ferroptosis. a) Analysis of PJA1 knockout (KO) on the binding of K48‐ and K63‐linked ubiquitination of GLO1 protein in the indicated PANC1 cells treated with erastin (5 µ m ) or RSL3 (500 n m ) for 6 h. b) Western blot analysis of GLO1 protein expression in the indicated PJA1 ‐KO PANC1 cells with or without the expression of wild‐type (WT) or C598A mutant after treatment with erastin (5 µ m ) or RSL3 (500 n m ) for 24 h. c–e) Analysis of cell viability, intracellular MGO, and MDA concentrations in the indicated PANC1 cells treated with erastin (5 µ m ) or RSL3 (500 n m ) for 6–24 h. f) Western blot analysis of GLO1 protein expression in WT or PJA1 overexpressed (KI) HCT116 cells after treatment with erastin (5 µ m ) or RSL3 (500 n m ) for 24 h. g–i) Analysis of cell viability, intracellular MGO, and MDA concentrations in the indicated HCT116 cells treated with erastin (5 µ m ) or RSL3 (500 n m ) for 6–24 h. j) Analysis of the interaction between GLO1 and PJA1, as well as GLO1 degradation, in GLO1 ‐KO PANC1 cells expressing either K47R/K48R or K157R/K159R GLO1 mutants, following treatment with erastin (5 µ m ) or RSL3 (500 n m ) for 24 h. k,l) Analysis of cell viability and intracellular MGO in the indicated PANC1 cells treated with erastin (5 µ m ) or RSL3 (500 n m ) for 6–24 h. All semi‐quantitative data are presented as means ± SD for n = 3 biologically independent samples (c–e,g–i,k,l). Statistical analyses were conducted using ANOVA with Tukey's multiple comparisons test (c–e,g–i,k,l).

Article Snippet: Plasmids containing the wild‐type PJA1 (OriGene, RC224490) and GLO1 (OriGene, RC203826) coding sequences served as templates for the mutagenesis reactions.

Techniques: Knock-Out, Binding Assay, Ubiquitin Proteomics, Western Blot, Expressing, Mutagenesis

Journal: Advanced Science

Article Title: Targeting Methylglyoxal Metabolism to Enhance Ferroptosis Sensitivity in Tumor Therapy

doi: 10.1002/advs.202505356

Figure Lengend Snippet: Targeting MPO pathway enhances ferroptosis sensitivity in xenograft models. a) Schematic of the experimental setup showing subcutaneous implantation of human PANC1 or HCT116 cells in immunocompromised NOD‐SCID mice, followed by a treatment protocol over 3 weeks. b) Growth curves for tumors from subcutaneously implanted the indicated PANC1 or HCT116 cells in NOD‐SCID mice (n = 5 per group) following the treatment described in (a). c–f) Analysis of GLO1, MGO, and MDA levels in isolated tumors on day 31 (n = 5 mice per group). Bar = 100 µm. g) Schematic of the experimental protocol for subcutaneous implantation of PDXs in NOD‐SCID mice, including a 3‐week treatment regimen. h) Western blot analysis showing GLO1 expression levels in PDAC tumors categorized by high or low expression. i) Tumor growth curves for PDXs subcutaneously implanted in NOD‐SCID mice (n = 5 per group). Data are presented as means ± SD, with statistical analysis performed using ANOVA (b,d–f,i).

Article Snippet: Plasmids containing the wild‐type PJA1 (OriGene, RC224490) and GLO1 (OriGene, RC203826) coding sequences served as templates for the mutagenesis reactions.

Techniques: Isolation, Western Blot, Expressing

Journal: Advanced Science

Article Title: Targeting Methylglyoxal Metabolism to Enhance Ferroptosis Sensitivity in Tumor Therapy

doi: 10.1002/advs.202505356

Figure Lengend Snippet: Targeting MPO pathway enhances ferroptosis sensitivity in orthotopic and xenograft models. a) Schematic diagram of the experimental setup: orthotopic implantation of mouse KPC cells in C57BL/6J mice, followed by a 3‐week treatment protocol. b) Survival curves for mice treated with vehicle, IKE, and/or BrBzGCp2 (n = 20 mice per group). c–e) Analysis of MGO, MDA, and GLO1 levels in isolated tumors on day 28 (n = 5 mice per group). f) IHC analysis of CD8 and CD4 in isolated tumors on day 28 (n = 5 mice per group). Bar = 100 µm. g) ELISA analysis of serum cytokines on day 28 (n = 5 mice per group). h) Schematic of the experimental setup showing subcutaneous implantation of human PANC1 or HCT116 cells in immunocompromised NOD‐SCID mice, followed by a treatment protocol over 3 weeks. i) Growth curves for tumors from subcutaneously implanted the indicated PANC1 or HCT116 cells in NOD‐SCID mice (n = 5 per group) following the treatment described in (h). j) Growth curves for tumors from subcutaneously implanted the indicated WT and GLO1 ‐KO PANC1 or HCT116 cells in NOD‐SCID mice (n = 5 per group) following the treatment with IKE. Data are presented as means ± SD. Statistical analyses were conducted using ANOVA for panels c–g, i, and j, with log‐rank tests for panel B.

Article Snippet: Plasmids containing the wild‐type PJA1 (OriGene, RC224490) and GLO1 (OriGene, RC203826) coding sequences served as templates for the mutagenesis reactions.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Targeting Methylglyoxal Metabolism to Enhance Ferroptosis Sensitivity in Tumor Therapy

doi: 10.1002/advs.202505356

Figure Lengend Snippet: Clinical significance of PJA1 and GLO1 expression in PDAC patients. a) Analysis of PJA1 and GLO1 expression and localization in pancreatic tumors using The Human Protein Atlas. Bar = 100 µm. b,c) Correlation of PJA1 or GLO1 expression with survival in PDAC patients using The Cancer Genome Atlas. The selected cut‐off was the optimal choice. d) A model illustrating PJA1‐mediated GLO1 proteasomal degradation, leading to ferroptosis via MGO accumulation and subsequent autophagic degradation of ferritin and GPX4.

Article Snippet: Plasmids containing the wild‐type PJA1 (OriGene, RC224490) and GLO1 (OriGene, RC203826) coding sequences served as templates for the mutagenesis reactions.

Techniques: Expressing

Journal: Cell chemical biology

Article Title: Chemoproteomic Profiling of a Pharmacophore-Focused Chemical Library.

doi: 10.1016/j.chembiol.2020.04.007

Figure Lengend Snippet: Figure 2. Discovery of Glo1-Binding Com- pounds 7oa3 and 7qc8 (A) Fluorescence gel images of HEK293 cells co- treated with probe 1 (5 mM) and various test pools (eight compounds each, ~240 mM): 7o2-7o11 test pools (left gel), 7q2-7q11 test pools (right gel). The loss of the ~25-kDa band (black arrow) was observed in the presence of test pools, 7o3 and 7q8. (B) Fluorescence gel images of HEK293 cells co- treated with probe 1 (5 mM) and the individual compounds (30 mM) of 7o3 (left gel) and 7q8 (right gel). (C) Chemical structures of the screening hits, 7oa3 and 7qc8. (D) Silver-stained gel image of proteins enriched by biotin pulldown of probe 1. The ~25-kDa band (black arrow) was identified as Glo1. (E) Western blot analysis showed the enrichment of Glo1 by probe 1 (5 mM), which was abolished in the presence of 7oa3 or 7qc8 (50 mM).

Article Snippet: The membrane was blocked for 1 h with 5% skim milk and incubated overnight with mouse anti-glo1 antibody (Novus Biologicals, 1:200) in blocking buffer.

Techniques: Binding Assay, Fluorescence, Staining, Western Blot

Journal: Cell chemical biology

Article Title: Chemoproteomic Profiling of a Pharmacophore-Focused Chemical Library.

doi: 10.1016/j.chembiol.2020.04.007

Figure Lengend Snippet: Figure 3. Glo1 Activity, Structure Optimization, and Protein Profiling of 7oa3 and 7qc8 Derivatives (A) Detoxification of methylglyoxal (MG) by the glyoxalase system. (B) The activity of recombinant human Glo1 in the presence of indomethacin, probe 1, 7oa3, and 7qc8 (5 mM each). Data represent average values ±SD (n = 3). (C) The structure of compounds 2–16. The modified groups are indicated in red. (D)The activity of recombinant human Glo1 in the presence of compounds 2–16 (5 mM). Data represent average values ±SD (n = 3).

Article Snippet: The membrane was blocked for 1 h with 5% skim milk and incubated overnight with mouse anti-glo1 antibody (Novus Biologicals, 1:200) in blocking buffer.

Techniques: Activity Assay, Recombinant

Journal: Cell chemical biology

Article Title: Chemoproteomic Profiling of a Pharmacophore-Focused Chemical Library.

doi: 10.1016/j.chembiol.2020.04.007

Figure Lengend Snippet: Figure 5. Molecule 9 Lies at a Hydrophobic Site Abutting the Substrate Binding Site (A) The 2Fo Fc electron density map of molecule 9 contoured at 1.0 sigma. (B) The surface protein structure of the Glo1 homodimer-9 complex. The Glo1 monomers, represented in cyan and green colors, create two active sites situated at the dimer interface. Molecule 9 lies at the border of the substrate binding site, wherein the tetrahydropyridoindole ring of 9 sits on the hydrophobic region formed by Phe163, Val150, and Trp171, while the oxygen atom of the trifluoromethoxy phenyl group is located within the H-bond distance (3.0 A˚ ) of the guanidinium side chain of Arg123. (C) Comparison of mGLO1-9 complex (PDB: 6L0U) and superimposed hGLO1-HIPC-GSH complex (PDB: 1QIN). Molecule 9 and HIPC-GSH molecules are shown as stick models in magentas and gray, respectively. The carbon atoms of the protein in the mGLO1-9 complex are drawn in green while those of hGLO1- HIPC-GSH complex are in gray. (D and E) Kinetic analysis of 9 against mGlo1. (D) Dixon plot of initial velocity versus varying 9 concentrations. (E) Lineweaver-Burk plot of initial velocity versus varying substrate, MG-SG concentrations. Data represent average values ± SD (n = 3). (F) The activity of wild-type (wt) and mGlo1 mutants: F163G, R123G, and K156G in the presence of increasing concentration of 9 (0, 1, 2, 5, and 10 mM). Data represent average values ± SD (n = 3), *p = 0.01, **p = 0.007.

Article Snippet: The membrane was blocked for 1 h with 5% skim milk and incubated overnight with mouse anti-glo1 antibody (Novus Biologicals, 1:200) in blocking buffer.

Techniques: Binding Assay, Comparison, Activity Assay, Concentration Assay

Journal: Poultry Science

Article Title: Dynamic changes of protein lactylation and their correlations with the glycolytic process during the postmortem acidification of broiler breast

doi: 10.1016/j.psj.2024.104354

Figure Lengend Snippet: The suppliers and dilution ratios for the primary and secondary antibodies.

Article Snippet: GLO1 , Abclonal , 1:2000 , A4329.

Techniques:

Journal:

Article Title: Genetic Analysis of Lysosomal Trafficking in Caenorhabditis elegans

doi: 10.1091/mbc.E05-01-0060

Figure Lengend Snippet: Birefringent gut granule mislocalization in Glo mutants. Wild-type pretzel-stage embryos (A and B) and L1-stage larvae (C and D) contained birefringent gut granules (white arrows in B and white arrowheads in D) within intestinal cells. The intestinal lumen is marked with a black arrow in C and D. apt-6 (E and F) pretzel-stage embryos contained gut granules within the intestinal lumen (black arrow) and a reduced number of gut granules (white arrowheads) within intestinal cells. (G and H) The presence of birefringent material within the intestinal lumen (black arrow) and intestinal cells (white arrowheads) is shown at higher magnification in a newly hatched apt-6 larva. apt-7 (I and J) pretzel-stage embryos contained gut granules within the intestinal lumen (black arrow) and a reduced number of gut granules (white arrowheads) within intestinal cells. glo-1 (K and L), glo-2 (M and N), and glo-4 (Q and R) pretzel-stage embryos lacked gut granules within the intestinal cell cytoplasm and instead gut granules (black arrows) were localized in the intestinal lumen. glo-3 (O and P) pretzel stage embryos mislocalized birefringent granules to the intestinal lumen (black arrow) and typically contained between one and five birefringent gut granules (white arrowhead) within intestinal cells. In A, B, E, and F, and I-R intestinal cells are located between the black arrowheads. C. elegans embryos are ∼50 μm in length.

Article Snippet: Affinity-purified antibodies recognizing GLO-1 were generated by Bethyl Laboratories (Montgomery, TX).

Techniques:

Journal:

Article Title: Genetic Analysis of Lysosomal Trafficking in Caenorhabditis elegans

doi: 10.1091/mbc.E05-01-0060

Figure Lengend Snippet: Embryos require glo-1 for the assembly of acidified gut granules within the intestine. Intestinal cells in wild-type bean (A and B), 1.5-fold (C and D), and pretzel stage (E and F) embryos contained many acidic compartments stained by acridine orange. In wild type, the majority of gut granules were present within acidified compartments. (G–L) glo-1 intestinal cells at the same embryonic stages lacked birefringent gut granules and acidic structures stained by acridine orange. In all panels, the intestinal primordium is located between the black arrowheads. Acridine orange-stained apoptotic corpses anterior of the intestinal primordium in wild type (B and D) and glo-1 (H and J) embryos.

Article Snippet: Affinity-purified antibodies recognizing GLO-1 were generated by Bethyl Laboratories (Montgomery, TX).

Techniques: Staining

Journal:

Article Title: Genetic Analysis of Lysosomal Trafficking in Caenorhabditis elegans

doi: 10.1091/mbc.E05-01-0060

Figure Lengend Snippet: Analysis of autofluorescent and acidified gut granules in young adult-stage Glo mutants. (A–C) Wild-type animals contained numerous autofluorescent and LysoTracker Red-stained acidified gut granules within intestinal cells, visualized in fluorescein isothiocyanate and rhodamine channels, respectively. Intestinal cells in young adult apt-6 (D–F), apt-7 (G–I), vps-16 (J–L), glo-1 (P–R), glo-2 (S–U), and glo-4 (Y–A′) animals contained few or no autofluorescent and acidified gut granules. vps-41 (M–O) animals contained a reduced number of gut granules that were localized in intestinal cells near the basal membrane. Reduced numbers of gut granules were present within the intestinal cells of glo-3 (V–X) animals. Posterior glo-3 intestinal cells (shown in V–X) contained on average 2 to 3 times the number of autofluorescent and acidic gut granules as anterior intestinal cells. In all panels the intestinal lumen (apical) is marked with a black arrow.

Article Snippet: Affinity-purified antibodies recognizing GLO-1 were generated by Bethyl Laboratories (Montgomery, TX).

Techniques: Staining

Journal:

Article Title: Genetic Analysis of Lysosomal Trafficking in Caenorhabditis elegans

doi: 10.1091/mbc.E05-01-0060

Figure Lengend Snippet: Elongation of wild-type and Glo mutant embryos. Images of wild-type (A–C) and glo-1 (D–F) embryos were captured at the times listed after first cell cleavage. glo-1 embryos elongated normally and reached the fourfold stage (E and G), however soon after they reduced to 3-fold in length (F and G) and displayed ripples in their epidermis/cuticle (F, black arrows). glo-2 (H) and glo-3 (I) embryos similarly elongated to fourfold before reducing to 3-fold length. (G–I) Glo embryos lacked birefringent gut granules before they occurred in the intestinal lumen (arrow denotes average time of occurrence). In G–I, the length of individual embryos and the presence/localization of birefringent material was monitored every 45 min starting at the 1.5-fold stage. Bars represent 95% confidence interval.

Article Snippet: Affinity-purified antibodies recognizing GLO-1 were generated by Bethyl Laboratories (Montgomery, TX).

Techniques: Mutagenesis

Journal:

Article Title: Genetic Analysis of Lysosomal Trafficking in Caenorhabditis elegans

doi: 10.1091/mbc.E05-01-0060

Figure Lengend Snippet: glo-1 is necessary for the formation of acidified and terminal endocytic organelles in larvae and adults. In wild type, gut granules were weakly autofluorescent in the rhodamine channel (B). After staining with dyes that label acidic (D) or terminal endocytic (F and H) compartments, gut granules in wild type accumulated the dyes and displayed much stronger fluorescence. In contrast, intestinal cells in glo-1 mutants did not stain with acidic (P) or terminal the endocytic (R) organelle marker TRITC-bovine serum albumin. In 25% (n = 20) of glo-1 animals, the endocytic marker FM4-64 accumulated in organelles localized near the apical surface of intestinal cells (T). In A–H and M–T, the intestinal lumen is marked with a black arrow. glo-1 mutants did not have defects in receptor mediated endocytosis (compare J and V) of YP170::GFP into oocytes (white arrowheads mark the most proximal oocytes). Fluid phase endocytosis of GFP localized within the body cavity by coelomocytes (white circle marks cell periphery) was not disrupted in glo-1 mutants (compare L and X).

Article Snippet: Affinity-purified antibodies recognizing GLO-1 were generated by Bethyl Laboratories (Montgomery, TX).

Techniques: Staining, Fluorescence, Marker

Journal:

Article Title: Genetic Analysis of Lysosomal Trafficking in Caenorhabditis elegans

doi: 10.1091/mbc.E05-01-0060

Figure Lengend Snippet: Subcellular localization of GLO-1. Wild-type (A and B) and glo-1(zu437) (C and D) E8 stage embryos stained with anti-GLO-1 antibodies. (E–H) glo-1(zu437) strains carrying an extrachromosomal transgene containing the glo-1 promoter driving the expression of GLO-1::GFP in intestinal precursor cells at the E4 (E and F) and E8 (G and H) stages. glo-1(zu437); GLO-1::GFP embryos at the E8 stage (I–L) and 1.5-fold stage (M-P) after staining with anti-GFP and anti-VHA-17/FUS-1 antibodies. GLO-1::GFP and FUS-1 colocalized to the same intestinal organelles (white arrows). (A–L) Black asterisks marks intestinal nuclei. (M–P) Black arrowheads mark the anterior of the intestine. (R–T) High magnification of the intestine of a 1.5-fold, glo-1(zu437); GLO-1::GFP embryo (boxed region in Q). Most gut granules, pseudocolored red in S and T, are present within vesicles containing GLO-1::GFP (R and T) (white arrows). Some gut granules are not present within GLO-1::GFP containing organelles (white arrowhead).

Article Snippet: Affinity-purified antibodies recognizing GLO-1 were generated by Bethyl Laboratories (Montgomery, TX).

Techniques: Staining, Expressing

Journal:

Article Title: Genetic Analysis of Lysosomal Trafficking in Caenorhabditis elegans

doi: 10.1091/mbc.E05-01-0060

Figure Lengend Snippet: Analysis of endo-lysosomal organelles in glo-1 embryos. Antibodies that recognize the V-ATPase subunits VHA-17/FUS-1 (A and B) and VHA-11 (C and D) stained organelles in wild-type 1.5-fold (A) and bean-stage (C) embryos. These organelles were absent glo-1(–) embryos at similar stages (B and D). Anti-GFP antibodies used to stain embryos expressing RME-1 (E and F), RAB-5 (G and H) LMP-1 (I and J), and RME-8 (K and L) GFP protein fusions revealed a similar staining pattern in wild-type and glo-1(–) pretzel-stage embryos, with the exception that LMP-1::GFP structures were slightly enlarged in glo-1 embryos (white arrow in J). In all panels, intestinal cells are located between the black arrowheads.

Article Snippet: Affinity-purified antibodies recognizing GLO-1 were generated by Bethyl Laboratories (Montgomery, TX).

Techniques: Staining, Expressing

Journal:

Article Title: Genetic Analysis of Lysosomal Trafficking in Caenorhabditis elegans

doi: 10.1091/mbc.E05-01-0060

Figure Lengend Snippet: Predicted structure of the glo-1 gene and encoded protein. (A) The intron-exon structure of glo-1 and location of the glo-1 mutations are shown. (B) The predicted C. elegans GLO-1 protein sequence (AAY42967) is aligned to the C. briggsae GLO-1 (CBP14321 and CBP14320), D. melanogaster RabRP1 (AB035646), Dictyostelium RabE (AF116859), human Rab38 (NP071732), and human Rab32 (Q13637) proteins. Positions of GTPase (G1-G5), Rab family (F1–F5), and prenylation motifs are shown.

Article Snippet: Affinity-purified antibodies recognizing GLO-1 were generated by Bethyl Laboratories (Montgomery, TX).

Techniques: Sequencing

Journal:

Article Title: Genetic Analysis of Lysosomal Trafficking in Caenorhabditis elegans

doi: 10.1091/mbc.E05-01-0060

Figure Lengend Snippet: Expression of glo-1 in embryos and adults. (A–F) Wild-type strains carried an extrachromosomal transgene containing the glo-1 promoter driving the expression of gfp (glo-1::gfp). gfp expression in intestinal precursors at the E2 (A and B) and 1.5-fold (C and D) stages are shown. (A and B) Nuclei of intestinal precursors are marked with asterisks. (C and D) Black arrowheads denote the anterior of the intestinal primordium. (E and F) GFP was expressed within the adult intestine (black arrow marks lumen). Bar (E and F), 25 μm.

Article Snippet: Affinity-purified antibodies recognizing GLO-1 were generated by Bethyl Laboratories (Montgomery, TX).

Techniques: Expressing