Journal: Clinical and Experimental Immunology

Article Title: The effects of maternal anti-alpha-enolase antibody expression on the brain development in offspring

doi: 10.1093/cei/uxac086

Figure Lengend Snippet: research design. Each female mouse was immunized twice with 100 μg recombinant ENO1 protein in Freund’s adjuvant to establish a mouse model with a high serum anti-ENO1 antibody (H-ENO1Ab) level. The control female mice were only s.c. injected with an equivalent volume of PBS in replacement of ENO1 protein. Some of the pups were examined for learning and memory abilities using the Morris water maze (MWM) test before they were sacrificed on P40.

Article Snippet: After blocking with 5% BSA, the slices were incubated with the following primary antibodies at 4°C overnight: rabbit antibodies against ENO1 (1:500, BIOSS, China), NeuN (1:500, Abcam, MA), rat antibodies against CD34 (1:50, Abcam), rabbit anti-C3 antibodies (1:50, Abcam), rabbit anti-CD16 antibodies (1:200, BIOSS), mouse anti-C5b-9 antibodies (1:300, CST, MA), and rabbit anti-cleaved caspase-3 (1:400, CST).

Techniques: Recombinant, Injection

Journal: Clinical and Experimental Immunology

Article Title: The effects of maternal anti-alpha-enolase antibody expression on the brain development in offspring

doi: 10.1093/cei/uxac086

Figure Lengend Snippet: the levels of ENO1Ab in the H-ENO1Ab dams and their pups. Serum and amniotic fluid samples were collected as depicted in . Anti-ENO1 total IgG and its subtypes were measured by enzyme-linked immunosorbent assay (ELISA). The results are shown above for the dams (A, n = 6-14/group, ** P < 0.01, *** P < 0.001 vs. the control group) and the pups (B, n = 14-19 for total IgG, n = 5-8 for IgG1, n = 5-9 for IgG2a, IgG2b and IgG3, ** P < 0.01, *** P < 0.001, not significantly (NS) vs. the pups of the control group).

Article Snippet: After blocking with 5% BSA, the slices were incubated with the following primary antibodies at 4°C overnight: rabbit antibodies against ENO1 (1:500, BIOSS, China), NeuN (1:500, Abcam, MA), rat antibodies against CD34 (1:50, Abcam), rabbit anti-C3 antibodies (1:50, Abcam), rabbit anti-CD16 antibodies (1:200, BIOSS), mouse anti-C5b-9 antibodies (1:300, CST, MA), and rabbit anti-cleaved caspase-3 (1:400, CST).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Clinical and Experimental Immunology

Article Title: The effects of maternal anti-alpha-enolase antibody expression on the brain development in offspring

doi: 10.1093/cei/uxac086

Figure Lengend Snippet: detection of ENO1 protein expression in the brain tissues of the pups by western blot. One intact female mouse was mated with one male mouse, and the brain tissues of the pups were obtained for the detection of ENO1 protein expression on E13, P10, P21, and P40.

Article Snippet: After blocking with 5% BSA, the slices were incubated with the following primary antibodies at 4°C overnight: rabbit antibodies against ENO1 (1:500, BIOSS, China), NeuN (1:500, Abcam, MA), rat antibodies against CD34 (1:50, Abcam), rabbit anti-C3 antibodies (1:50, Abcam), rabbit anti-CD16 antibodies (1:200, BIOSS), mouse anti-C5b-9 antibodies (1:300, CST, MA), and rabbit anti-cleaved caspase-3 (1:400, CST).

Techniques: Expressing, Western Blot

Journal: Clinical and Experimental Immunology

Article Title: The effects of maternal anti-alpha-enolase antibody expression on the brain development in offspring

doi: 10.1093/cei/uxac086

Figure Lengend Snippet: IgG deposition and the distribution of C3, MAC, and CD16 in the brain tissue of the pups from H-ENO1Ab dams. All the pups were obtained as depicted in . (A) Representative images of IgG deposition in the brain tissues from the pups of H-ENO1Ab dams and those of the control group on P10 and P40 ( n = 4/group) after immunofluorescence (IF) staining with FITC-conjugated goat anti-mouse IgG (green, ×200). (B) Representative images for the colocalization of IgG and ENO1, NeuN, and CD34 in the brain tissues from the pups of H-ENO1Ab dams and those of the control group on P10 ( n = 4/group) after double IF staining with FITC-conjugated goat anti-mouse IgG (green) and rabbit anti-ENO1 and NeuN, rat anti-CD34 (red, ×200). (C and E) Representative images of the colocalization of IgG and C3 and CD16 in the brain tissues from the pups of H-ENO1Ab dams and those of the control group on P10 and P40 ( n = 4/group) after double IF staining with FITC-conjugated goat anti-mouse IgG (green) and rabbit anti-C3 and CD16 (red, ×200). (D) Representative images of MAC expression in the brain tissues from the pups of H-ENO1Ab dams and the those of the control group on P10 and P40 ( n = 4/group) after IF staining with mouse anti-C5b-9 (green, ×200). Nuclei were counterstained with DAPI (blue).

Article Snippet: After blocking with 5% BSA, the slices were incubated with the following primary antibodies at 4°C overnight: rabbit antibodies against ENO1 (1:500, BIOSS, China), NeuN (1:500, Abcam, MA), rat antibodies against CD34 (1:50, Abcam), rabbit anti-C3 antibodies (1:50, Abcam), rabbit anti-CD16 antibodies (1:200, BIOSS), mouse anti-C5b-9 antibodies (1:300, CST, MA), and rabbit anti-cleaved caspase-3 (1:400, CST).

Techniques: Immunofluorescence, Staining, Expressing

Journal: Theranostics

Article Title: Counterintuitive production of tumor-suppressive secretomes from Oct4- and c-Myc-overexpressing tumor cells and MSCs.

doi: 10.7150/thno.70549

Figure Lengend Snippet: Figure 7. Prediction of the tumor suppressors in CM by mass spectrometry-based whole-genome proteomics. The single and double asterisks indicate p < 0.05 and 0.01, respectively. CN = control, Oct4 = Oct4 plasmids, c-Myc = c-Myc plasmids, Sox2 = Sox2 plasmids, Klf4 = Klf4 plasmids, and CM = conditioned medium. (A) Summary list of the potential tumor suppressors by mass spectrometry-based whole-genome proteomics. (B) Enolase 1 (Eno1), Hsp90ab1 (HSP), Eef2, and vinculin (VCL) as 4 tumor-suppressor candidates based on MTT-based viability. (C) Upregulation of Eno1, Hsp90ab1, Eef2, VCL, p53, and Trail in 4T1.2 cell-derived CM with the overexpression of Oct4, c-Myc, and the treatment with OAC2. The overexpression of Sox2 and Klf4 did not alter their levels. (D) Alterations in the levels of Eno1 and Hsp90ab1 in Oct4 and c-Myc CM by ELISA. (E) Reduction in MTT-based viability of 4T1.2 cells by the treatment with Eno1 and/or Hsp90ab1 recombinant proteins. (F) Tumor selectivity from the MTT-based viability of tumor cells (4T1.2 mammary tumor cells, EO771 mammary tumor cells, and MDA-MB-231 breast cancer cells) and human epithelium cells (KTB34-hTERT and KTB22-hTERT). Tumor selectivity is defined as a ratio of (MTT-based reduction in tumor cells) to (MTT-based reduction in non-tumor cells).

Article Snippet: Western blotting was conducted using antibodies against Eno1 and CD44 (Cell Signaling).

Techniques: Mass Spectrometry, Control, Derivative Assay, Over Expression, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Theranostics

Article Title: Counterintuitive production of tumor-suppressive secretomes from Oct4- and c-Myc-overexpressing tumor cells and MSCs.

doi: 10.7150/thno.70549

Figure Lengend Snippet: Figure 8. Tumor-promoting effects by the overexpression of Eno1, Eef2, and VCL in 4T1.2 mammary tumor cells, and tumor-suppressing effects by the administration of their recombinant proteins. The double asterisk indicates p < 0.01. CN = control, Eno1 = enolase 1, VCL = vinculin, and Hsp = Hsp90ab1. (A-C) Elevation in EdU-based proliferation, transwell invasion, and the upregulation of Lrp5, MMP9, Runx2, TGFβ, and Snail by the overexpression of Eno1, Eef2, and VCL in 4T1.2 tumor cells. (D) Decrease in EdU-based proliferation by the administration of Eno1, Eef2, and VCL recombinant proteins. (E) Reduction in transwell invasion by the administration of Eno1, Eef2, and VCL recombinant proteins. (F) Downregulation of Lrp5, MMP9, Runx2, TGFβ, and Snail in 4T1.2 tumor cells by the administration of Eno1, Eef2, and VCL recombinant proteins. (G) Co-immunoprecipitation of CD44 by Eno1 in 4T1.2 cells. (H-I) Suppression of the reduction in MTT-based viability by Eno1 in response to the silencing of CD44. (J) Downregulation of Lrp5, Runx2, MMP9 and Snail in 4T1.2 cells by the administration of Eno1 recombinant proteins, and the suppression by the silencing of CD44. (K-L) Western blotting of wild-type and mutant CD44 proteins, which were pulled down by Halo-tagged Eno1 proteins. PD = pull-down assay, NC = negative control, pl = Eno1 or CD44 proteins from plasmid transfection, MT = mutant CD44 without a cytoplasmic domain, and WT = wild-type CD44.

Article Snippet: Western blotting was conducted using antibodies against Eno1 and CD44 (Cell Signaling).

Techniques: Over Expression, Recombinant, Control, Immunoprecipitation, Western Blot, Mutagenesis, Pull Down Assay, Negative Control, Plasmid Preparation, Transfection

Journal: Theranostics

Article Title: Counterintuitive production of tumor-suppressive secretomes from Oct4- and c-Myc-overexpressing tumor cells and MSCs.

doi: 10.7150/thno.70549

Figure Lengend Snippet: Figure 9. Downregulation of PDL-1 and Kdm3a in 4T1.2 mammary tumor cells by Hsp90ab1, Eno1, Eef2, and VCL, and the suppression of the development of osteoclasts. CN = control, Hsp = Hsp90ab1, Eno1 = enolase 1, VCL = vinculin, Oct4 = Oct4 plasmids, and CM = conditioned medium. The double asterisk indicates p < 0.01. (A) Reduction in the size and weight of mammary tumors by the daily intravenous administration of 1 µg/mL Eno1. EO771 mammary tumor cells were inoculated into the mammary fat pad of C57BL/6 female mice (N = 10). (B) Reduction in p-AKT, NFkB p65, p-ERK, and TNFα in 4T1.2 cells in response to Eno1 recombinant proteins. (C) Reduction in PDL1 in 4T1.2 cells by Oct4/cMyc-overexpressing 4T1.2 CM, and Eno1, Hsp, Eef2, and VCL recombinant proteins. (D) Reduction in Kdm3a in 4T1.2 cells in response to Eno1, Hsp90ab1, Eef2, and/or VCL recombinant proteins. (E) Elevation of Kdm3a in 4T1.2 cells by the overexpression of Eno1, Eef2, and VCL. (F) Suppression of RANKL-stimulated osteoclast development by Oct4-overexpressing CM. (G) Reduction in the levels of cathepsin K and NFATc1 in RANKL-stimulated osteoclasts by Oct4-overexpressing CM. (H) Regulatory mechanism by the tumor-suppressive secretomes, which were derived from Oct4- and c-Myc-overexpressing tumor cells.

Article Snippet: Western blotting was conducted using antibodies against Eno1 and CD44 (Cell Signaling).

Techniques: Control, Recombinant, Over Expression, Derivative Assay

Journal: BMC Molecular Biology

Article Title: Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

doi: 10.1186/1471-2199-9-26

Figure Lengend Snippet: Identification of proteins in the secretome of hMADS cells at early step of adipogenesis and osteogenesis.

Article Snippet: Antibodies directed against ENO1, PEDF, BIGH3 and SPARC were supplied from Santa Cruz Biotechnology (Santa Cruz, CA), Chemicon International (Temecula, CA), R&D Systems (Minneapolis, MN) and Haematologic Technologies Inc. (Essex Junction, VT), respectively.

Techniques: Membrane, Binding Assay, Variant Assay, Control

Journal: BMC Molecular Biology

Article Title: Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

doi: 10.1186/1471-2199-9-26

Figure Lengend Snippet: Secretion levels of 8 candidates released from hMADS cells during the commitment to adipocytes and osteoblasts . The activity of MMP2 has been evaluated by gelatin zymography ( A ). The expression of SPARC ( B ), ENO1( C ), PEDF ( D ), GRP78 ( E ), BIGH3 ( F ), PTX3 ( G ) and PAI-1 ( H ) has been analyzed by Western blot. The bar graphs report the levels of expression of every single candidate as the mean of three independent experiments after 6 h of incubation. The values are indicated as arbitrary units. *: p < 0.05. Two μg of secreted proteins have been loaded for each gel.

Article Snippet: Antibodies directed against ENO1, PEDF, BIGH3 and SPARC were supplied from Santa Cruz Biotechnology (Santa Cruz, CA), Chemicon International (Temecula, CA), R&D Systems (Minneapolis, MN) and Haematologic Technologies Inc. (Essex Junction, VT), respectively.

Techniques: Activity Assay, Zymography, Expressing, Western Blot, Incubation

Journal: Diagnostics

Article Title: Alpha-Enolase (ENO1) Correlates with Invasiveness of Cutaneous Melanoma—An In Vitro and a Clinical Study

doi: 10.3390/diagnostics12020254

Figure Lengend Snippet: The expression level of ENO1 in the cell lysates from primary melanocytes and melanoma cell lines. Representative Western blots showing ENO1 and Akt 1/2/3 expression (for normalization) in protein lysates obtained from the primary human melanocytes (HEM) and indicated melanoma cell lines ( a ). Densitometric ENO1/Akt ratios are shown as mean values ( n = 3 except for HEM, n = 2) ± standard error of the mean (SEM) ( b ). The significance level was set at p = 0.001–0.0001 (***).

Article Snippet: Then, membranes were incubated for 1.5 h at room temperature (RT) with primary rabbit antibodies directed against ENO1 (PA5−13459 dilution 1:1000, Thermo Fisher, Waltham, MA, USA) and 1/2/3 Akt (sc−8312, H−136, dilution 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot

Journal: Diagnostics

Article Title: Alpha-Enolase (ENO1) Correlates with Invasiveness of Cutaneous Melanoma—An In Vitro and a Clinical Study

doi: 10.3390/diagnostics12020254

Figure Lengend Snippet: Enolase expression in melanoma cell lines determined by indirect immunofluorescence and confocal microscopy. ( a ) Single optical sections showing cells stained for ENO1 (green) and DAPI (blue). In the second column ENO1 signal was enhanced by brightness adjustment for the sake of better visualization. Raw images (shown in the last column) were subjected to fluorescence signal intensity analysis. Bar—15 μm. ( b ) Fluorescence signal intensity of the ENO1 presented as a mean ± standard deviation. The significance level was set at p = 0.05–0.01 (*). Subsequent number of cells were analyzed: A375—40 cells, WM1341D—71 cells, WM9—25 cells, Hs294T—45 cells.

Article Snippet: Then, membranes were incubated for 1.5 h at room temperature (RT) with primary rabbit antibodies directed against ENO1 (PA5−13459 dilution 1:1000, Thermo Fisher, Waltham, MA, USA) and 1/2/3 Akt (sc−8312, H−136, dilution 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Immunofluorescence, Confocal Microscopy, Staining, Fluorescence, Standard Deviation

Journal: Diagnostics

Article Title: Alpha-Enolase (ENO1) Correlates with Invasiveness of Cutaneous Melanoma—An In Vitro and a Clinical Study

doi: 10.3390/diagnostics12020254

Figure Lengend Snippet: The enolase activity in protein lysates prepared from A375, Hs294T, WM1341D, and WM9 melanoma cells cultured under normoxia or hypoxia. Bars represent mean values ( n = 4) ± standard error of the mean (SEM). The significance level was set at p = 0.05–0.01 (*), p = 0.01–0.001 (**).

Article Snippet: Then, membranes were incubated for 1.5 h at room temperature (RT) with primary rabbit antibodies directed against ENO1 (PA5−13459 dilution 1:1000, Thermo Fisher, Waltham, MA, USA) and 1/2/3 Akt (sc−8312, H−136, dilution 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Activity Assay, Cell Culture

Journal: Diagnostics

Article Title: Alpha-Enolase (ENO1) Correlates with Invasiveness of Cutaneous Melanoma—An In Vitro and a Clinical Study

doi: 10.3390/diagnostics12020254

Figure Lengend Snippet: Representative results of immunohistochemical analysis of ENO1 expression in cutaneous melanoma patients. Low cytoplasmic ENO1 immunoreactivity in melanoma cells (( a ), 200×; ( b ), 400×). High expression of ENO1 in tumoral cells (( c ), 200×; ( d ), 400×).

Article Snippet: Then, membranes were incubated for 1.5 h at room temperature (RT) with primary rabbit antibodies directed against ENO1 (PA5−13459 dilution 1:1000, Thermo Fisher, Waltham, MA, USA) and 1/2/3 Akt (sc−8312, H−136, dilution 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Immunohistochemical staining, Expressing

Journal: Diagnostics

Article Title: Alpha-Enolase (ENO1) Correlates with Invasiveness of Cutaneous Melanoma—An In Vitro and a Clinical Study

doi: 10.3390/diagnostics12020254

Figure Lengend Snippet: Correlations between ENO1 expression and clinical parameters of cutaneous melanoma patients.

Article Snippet: Then, membranes were incubated for 1.5 h at room temperature (RT) with primary rabbit antibodies directed against ENO1 (PA5−13459 dilution 1:1000, Thermo Fisher, Waltham, MA, USA) and 1/2/3 Akt (sc−8312, H−136, dilution 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Diagnostics

Article Title: Alpha-Enolase (ENO1) Correlates with Invasiveness of Cutaneous Melanoma—An In Vitro and a Clinical Study

doi: 10.3390/diagnostics12020254

Figure Lengend Snippet: ENO1 expression and AJCC (American Joint Committee on Cancer) staging. The lowest ENO1 expression in tumoral cells was observed in patients with stage I cutaneous melanoma. In stages II-IV, it was observed a significant increasing of ENO1 immunoreactivity in neoplastic cells.

Article Snippet: Then, membranes were incubated for 1.5 h at room temperature (RT) with primary rabbit antibodies directed against ENO1 (PA5−13459 dilution 1:1000, Thermo Fisher, Waltham, MA, USA) and 1/2/3 Akt (sc−8312, H−136, dilution 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Diagnostics

Article Title: Alpha-Enolase (ENO1) Correlates with Invasiveness of Cutaneous Melanoma—An In Vitro and a Clinical Study

doi: 10.3390/diagnostics12020254

Figure Lengend Snippet: Correlations between ENO1 expression and histopathological parameters in primary tumors of cutaneous melanoma patients.

Article Snippet: Then, membranes were incubated for 1.5 h at room temperature (RT) with primary rabbit antibodies directed against ENO1 (PA5−13459 dilution 1:1000, Thermo Fisher, Waltham, MA, USA) and 1/2/3 Akt (sc−8312, H−136, dilution 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Diagnostics

Article Title: Alpha-Enolase (ENO1) Correlates with Invasiveness of Cutaneous Melanoma—An In Vitro and a Clinical Study

doi: 10.3390/diagnostics12020254

Figure Lengend Snippet: Kaplan–Meier analysis of the prognostic significance of ENO1 expression in cutaneous melanoma patients. Overexpression of ENO1 correlated with shorter cancer-specific overall survival ( a ) and shorter disease-free survival ( b ). p levels of the log-rank test.

Article Snippet: Then, membranes were incubated for 1.5 h at room temperature (RT) with primary rabbit antibodies directed against ENO1 (PA5−13459 dilution 1:1000, Thermo Fisher, Waltham, MA, USA) and 1/2/3 Akt (sc−8312, H−136, dilution 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Over Expression

Journal: Diagnostics

Article Title: Alpha-Enolase (ENO1) Correlates with Invasiveness of Cutaneous Melanoma—An In Vitro and a Clinical Study

doi: 10.3390/diagnostics12020254

Figure Lengend Snippet: Univariate Cox proportional hazards model.

Article Snippet: Then, membranes were incubated for 1.5 h at room temperature (RT) with primary rabbit antibodies directed against ENO1 (PA5−13459 dilution 1:1000, Thermo Fisher, Waltham, MA, USA) and 1/2/3 Akt (sc−8312, H−136, dilution 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques:

Journal: BMC Molecular Biology

Article Title: Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

doi: 10.1186/1471-2199-9-26

Figure Lengend Snippet: Identification of proteins in the secretome of hMADS cells at early step of adipogenesis and osteogenesis.

Article Snippet: Antibodies directed against ENO1, PEDF, BIGH3 and SPARC were supplied from Santa Cruz Biotechnology (Santa Cruz, CA), Chemicon International (Temecula, CA), R&D Systems (Minneapolis, MN) and Haematologic Technologies Inc. (Essex Junction, VT), respectively.

Techniques: Membrane, Binding Assay, Variant Assay, Control

Journal: BMC Molecular Biology

Article Title: Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

doi: 10.1186/1471-2199-9-26

Figure Lengend Snippet: Secretion levels of 8 candidates released from hMADS cells during the commitment to adipocytes and osteoblasts . The activity of MMP2 has been evaluated by gelatin zymography ( A ). The expression of SPARC ( B ), ENO1( C ), PEDF ( D ), GRP78 ( E ), BIGH3 ( F ), PTX3 ( G ) and PAI-1 ( H ) has been analyzed by Western blot. The bar graphs report the levels of expression of every single candidate as the mean of three independent experiments after 6 h of incubation. The values are indicated as arbitrary units. *: p < 0.05. Two μg of secreted proteins have been loaded for each gel.

Article Snippet: Antibodies directed against ENO1, PEDF, BIGH3 and SPARC were supplied from Santa Cruz Biotechnology (Santa Cruz, CA), Chemicon International (Temecula, CA), R&D Systems (Minneapolis, MN) and Haematologic Technologies Inc. (Essex Junction, VT), respectively.

Techniques: Activity Assay, Zymography, Expressing, Western Blot, Incubation

Journal: The Journal of Biological Chemistry

Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection

doi: 10.1074/jbc.M117.810440

Figure Lengend Snippet: Recombinant enolase1 from C. albicans has TGase activity. The C. albicans ENO1 gene was cloned in the pCold II plasmid and transformed into E. coli BL21 (DE3) pLysS-competent cells; protein production was induced at 23 °C for 24 h. A , Eno1 protein was purified by IMAC with a Ni 2+ -NTA–agarose column in native conditions as described, and elution fractions were evaluated by 12% SDS-PAGE; MWM , protein molecular weight markers. Empty vector was also transformed in bacteria and passed through the same IMAC column, and the fractions obtained were also analyzed as a negative control (data not shown). B , Western blot of purified recombinant protein using anti-His–tag polyclonal antibodies ( lane 2 ) and rabbit anti-rCaEno1 protein ( lane 3 ). C , Western blot of C. albicans cell fractions using anti-rCaEno1 polyclonal antibodies. WPE , whole-protein extracts; CW , cell wall fraction; MMF , mixed membrane fraction; S-35K , soluble cytosolic fraction. Arrows indicate Eno1 protein. D , enolase activity was determined with purified rCaEno1 protein. E , TGase activity determined with purified rCaEno1 protein. These results allowed us to conclude that rCaEno1 protein has both enolase and transglutaminase activities. Statistical unpaired t test. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001. Bars are shown with standard error of mean. E1, enolase 1; S-Ab, specific antibodies (anti-rCaEno1); Us-Ab, unspecific antibodies (anti-rEhPCNA); Cys, cystamine.

Article Snippet: Grids with the sections were floated on drops of the following solutions: ( a ) 0.1% skim milk and PBS-T (PBS and 0.05% Tween 20) for 30 min to diminish non-specific labeling; ( b ) rabbit antibody against Eno1 recombinant protein at a 1:20 dilution in PBS-T for 1 h at room temperature and overnight at 4 °C, and ( c ) goat anti-rabbit polyclonal antibody coupled to 10-nm gold particles for 2 h at room temperature (Zymed Laboratories Inc. and Thermo Fisher Scientific) (1:40 dilution in PBS-T).

Techniques: Recombinant, Activity Assay, Clone Assay, Plasmid Preparation, Transformation Assay, Purification, SDS Page, Molecular Weight, Bacteria, Negative Control, Western Blot, Membrane

Journal: The Journal of Biological Chemistry

Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection

doi: 10.1074/jbc.M117.810440

Figure Lengend Snippet: IEM localization of ENO1 in C. albicans . Micrographs A–C and D–F correspond to low and high magnifications of two sets of yeasts processed for immuno-gold labeling for detection of ENO1. G corresponds to a yeast processed to preserve general ultrastructure. H corresponds to the negative control incubated with normal rabbit serum. Scale bar, 0.2 μm.

Article Snippet: Grids with the sections were floated on drops of the following solutions: ( a ) 0.1% skim milk and PBS-T (PBS and 0.05% Tween 20) for 30 min to diminish non-specific labeling; ( b ) rabbit antibody against Eno1 recombinant protein at a 1:20 dilution in PBS-T for 1 h at room temperature and overnight at 4 °C, and ( c ) goat anti-rabbit polyclonal antibody coupled to 10-nm gold particles for 2 h at room temperature (Zymed Laboratories Inc. and Thermo Fisher Scientific) (1:40 dilution in PBS-T).

Techniques: Labeling, Negative Control, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Proteomic Study Identifies Glycolytic and Inflammation Pathways Involved in Recurrent Otitis Media

doi: 10.3390/ijms21239291

Figure Lengend Snippet: Dysregulated proteins identified by mass spectrometry in RAOM compared to ATH.

Article Snippet: After protein transfer the membrane was blocked by treatment with 5% defatted milk in TBS-tween 20 and incubated overnight at 4 °C with 1:700 diluted primary rabbit polyclonal antibody against Transitional endoplasmic reticulum ATPase (VCP (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)), and with 1:800 diluted primary rabbit polyclonal antibody against Alpha-enolase (ENO1 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)).

Techniques: Mass Spectrometry, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Proteomic Study Identifies Glycolytic and Inflammation Pathways Involved in Recurrent Otitis Media

doi: 10.3390/ijms21239291

Figure Lengend Snippet: Representative Western blotting analysis of VCP and ENO1 in ATH and RAOM. The intensities of the immunostained bands were normalized with the protein intensities measured by Red Ponceau from the same blot. The bar graph shows the relative quantitation (band density) of VCP and ENO1 in ATH and RAOM. Results are shown as a histogram (* indicates p < 0.05, while ** indicates p < 0.01 statistical difference) and each bar represents mean ± standard error.

Article Snippet: After protein transfer the membrane was blocked by treatment with 5% defatted milk in TBS-tween 20 and incubated overnight at 4 °C with 1:700 diluted primary rabbit polyclonal antibody against Transitional endoplasmic reticulum ATPase (VCP (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)), and with 1:800 diluted primary rabbit polyclonal antibody against Alpha-enolase (ENO1 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)).

Techniques: Western Blot, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: Proteomic Study Identifies Glycolytic and Inflammation Pathways Involved in Recurrent Otitis Media

doi: 10.3390/ijms21239291

Figure Lengend Snippet: Schematic diagram displaying putative pathogenic mechanisms In ROAM (left side): bacterial infections (1) triggers neutrophils degranulation (2), but this is not effective due to lower PKM and HBB levels (black arrow downwards). Defective bacterial clearing (3) causes chronic infection (through formation of biofilm) in the median ear, with a consequent increase of NETs formation (4), due to higher proteasome activity provoked by increased VCP and CAP1 levels (black arrow upwards). Chronic bacterial infection of ear tissue (5) (red) establishes a milieu in which recurrent infections (6) are facilitated. In ATH (right side), TH-17 cells are activated (1) and switch to glycolytic metabolism (gray square), with increased levels of ALDOC and ENO1 (black arrow upwards). Activation of these cells causes increasing Interleukin-17 (IL-17) (2) production. This cytokine (3) could drive epithelial expression of granulopoietic and chemotactic factors such as Interleukin-8 (IL-8), Granulocyte Colony-Stimulating Factor (G-CSF) and Macrophage Inflammatory Proteins (MIP) (big arrow downwards) that could induce swelling of adenoidal tissue (4) (green), followed by upper airway obstruction. The imperfect oxygen intake could then trigger a self-sustained cycle (5) (dotted arrow), inducing furthermore a switch toward glycolytic metabolism (created with BioRender.com).

Article Snippet: After protein transfer the membrane was blocked by treatment with 5% defatted milk in TBS-tween 20 and incubated overnight at 4 °C with 1:700 diluted primary rabbit polyclonal antibody against Transitional endoplasmic reticulum ATPase (VCP (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)), and with 1:800 diluted primary rabbit polyclonal antibody against Alpha-enolase (ENO1 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)).

Techniques: Infection, Activity Assay, Activation Assay, Expressing