rat monoclonal pe r d systems anti mouse gal3bp immunoblotting  (R&D Systems)

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Galectin-3-binding protein and future venous thromboembolism

doi: 10.1016/j.rpth.2023.102311

Figure Lengend Snippet: Distribution of baseline characteristics across quartiles of galectin-3-binding protein (gal3bp) plasma levels.

Article Snippet: Galectin-3-binding protein (gal3bp), a protein originally described as a tumor-associated antigen, interacts with various proteins, including gal3 [ ].

Techniques: Clinical Proteomics

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: Galectin-3-binding protein and future venous thromboembolism

doi: 10.1016/j.rpth.2023.102311

Figure Lengend Snippet: Plasma levels of galectin-3-binding protein (gal3bp) and venous thromboembolism (VTE). (A) Odds ratios (OR) with 95% CI for venous thromboembolism (VTE) according to quartiles of gal3bp plasma levels. Model 1, adjusted for age and sex; model 2, adjusted for age, sex, and body mass index (BMI); model 3, adjusted for age, sex, BMI, and high-sensitivity C-reactive protein. Women, quartile 1: <3.42 μg/mL; quartile 2: 3.42-4.82 μg/mL; quartile 3: 4.82-6.71 μg/mL; and quartile 4: ≥6.71 μg/mL. Men, quartile 1: <2.92 μg/mL; quartile 2: 2.92-3.96 μg/mL; quartile 3: 3.96-5.20 μg/mL; and quartile 4: ≥5.20 μg/mL. (B) Plots of estimated ORs for overall VTE as a function of time from blood sampling in Tromsø 4 (1994-1995) to VTE events. Participants with plasma levels of gal3bp in the highest quartile (Q4) were compared with those with gal3bp in the lowest quartile (Q1, reference category). Analyses were adjusted for age, sex, BMI, and high-sensitivity C-reactive protein. Risk estimates were not statistically significant ( P < .05). The number of VTE events is depicted above the plot. Note that because of a missing value in BMI, there were 414 VTE events when BMI was used as an adjustment variable.

Article Snippet: Galectin-3-binding protein (gal3bp), a protein originally described as a tumor-associated antigen, interacts with various proteins, including gal3 [ ].

Techniques: Clinical Proteomics, Binding Assay, Sampling

Journal: OncoImmunology

Article Title: TNBC-derived Gal3BP/Gal3 complex induces immunosuppression through CD45 receptor

doi: 10.1080/2162402x.2023.2246322

Figure Lengend Snippet: Figure 2. Expression of GalBP by TEX. a. Venn diagram depicting the percentage of 23 selected proteins revealed by mass spectrometry analysis that were expressed or contained by sEVs isolated from MDA-MB-231 and MCF10-A cells and in plasma of healthy volunteers and TNBC patients. Gal3BP as part of the 43.5% proteins expressed of the 23 selected proteins, was found in the samples from the TNBC groups. b. Bars showing the percentage of CD9 expression and CD9+/Gal3BP+ identified on sEVs isolated from normal breast cells (EX) or from TNBC cells (TEX). Results are expressed as mean±SD (n = 5); p values are shown in the figure (two-tailed t test). c. Representative dot blot showing double stained CD9+/Gal3BP+ in TEX vs EX by FACS analysis.

Article Snippet: The CD45 inhibitor (CAS 345,630-40-2, 2.9 μM; Calbiochem) is a PTPase CD45 inhibitor and a CD45 blocking peptide.19 Cells were transferred to FACS tubes and stained with antiCD3-PC5.5 (Beckman Coulter), anti-Gal3-PE (R&D Systems), anti-mouse Gal3BP (Novus Biologicals) and mouse antihuman FITC (Jackson ImmunoResearch).

Techniques: Expressing, Mass Spectrometry, Isolation, Clinical Proteomics, Two Tailed Test, Dot Blot, Staining

Journal: OncoImmunology

Article Title: TNBC-derived Gal3BP/Gal3 complex induces immunosuppression through CD45 receptor

doi: 10.1080/2162402x.2023.2246322

Figure Lengend Snippet: Figure 3. Formation of a complex of Gal3BP with Gal3. a. Gal3 secretion by MDA-MB-231, MCF10-A, ER+ MCF-7, and Her2+ BT474 cells. Results are expressed as mean ±SD (n = 3) (two-tailed t test). b. An assay was developed to evaluate the physical interactions between Gal3BP and Gal3. c. Bars showing a linear curve shape, depending on the sample dilution. Significantly higher physical interactions between Gal3BP and Gal3 derived from MDA-MB-231 and plasma of TNBC patients cells compared to MCF10-A or plasma of healthy women in triplicate (p < 0.007). Results are expressed as mean±SD (two-tailed t-test). d. Percentage of Gal3BP protein in TEX and EX immunoprecipitated with either anti-Gal3BP or anti-Gal3 antibodies (n = 3). Results are expressed as mean±SD; p values are shown in the figure (two-tailed t test). e. Percentage of Gal3 protein in TEX or EX immunoprecipitated with either anti Gal3BP or anti Gal3 antibodies (n = 3, mean±SD) (2-tailed t test). f. Western blot analysis showing expression of Gal3 only in TEX immunoprecipitated with anti-Gal3BP. Gal3BP expression was found in EX or TEX immunoprecipitated with anti-Ga3BP. Immunoprecipitation with anti-Gal3, resulted in higher Gal3BP expression in TEX than in EX. g. Immunofluorescence of PBMCs treated with EX. CD3 (purple) expressing Gal3BP (green)/Gal3 (red) complex in EX. Scale bar = 50 μm. H. Immunofluorescence of PBMCs treated with TEX. Triple staining of Gal3BP/Gal3 complex on CD3 cells (yellow). Pictures taken under confocal microscope, nucleus stained with DAPI (blue), Scale bar = 50 μm.

Article Snippet: The CD45 inhibitor (CAS 345,630-40-2, 2.9 μM; Calbiochem) is a PTPase CD45 inhibitor and a CD45 blocking peptide.19 Cells were transferred to FACS tubes and stained with antiCD3-PC5.5 (Beckman Coulter), anti-Gal3-PE (R&D Systems), anti-mouse Gal3BP (Novus Biologicals) and mouse antihuman FITC (Jackson ImmunoResearch).

Techniques: Two Tailed Test, Derivative Assay, Clinical Proteomics, Immunoprecipitation, Western Blot, Expressing, Immunofluorescence, Staining, Microscopy

Journal: OncoImmunology

Article Title: TNBC-derived Gal3BP/Gal3 complex induces immunosuppression through CD45 receptor

doi: 10.1080/2162402x.2023.2246322

Figure Lengend Snippet: Figure 4. Gal3/Gal3BP complex binding to CD45 and inhibiting CD45 signaling pathway in TEX. A. Percentage of T cells displaying Gal3BP/Gal3 complex with and without CD45 blocking in EX- and TEX-treated cells (n = 3). Results are expressed as mean±SD (two-tailed t test). B. Representative dot blot of FACS analysis of CD3+ gated cells expressing Gal3BP/Gal3 complex. C. Percentage of Lck Y505, Lck Y394, ZAP-70 Y493 and VAV-1 Y174 phosphorylation in PBMCs treated with EX, TEX, or complex (Gal3BP and Gal3 recombinant proteins). D. Representative histogram showing phosphorylation of proteins in the CD45 pathway after treatment with EX (red) or TEX (blue).

Article Snippet: The CD45 inhibitor (CAS 345,630-40-2, 2.9 μM; Calbiochem) is a PTPase CD45 inhibitor and a CD45 blocking peptide.19 Cells were transferred to FACS tubes and stained with antiCD3-PC5.5 (Beckman Coulter), anti-Gal3-PE (R&D Systems), anti-mouse Gal3BP (Novus Biologicals) and mouse antihuman FITC (Jackson ImmunoResearch).

Techniques: Binding Assay, Blocking Assay, Two Tailed Test, Dot Blot, Expressing, Phospho-proteomics, Recombinant

Journal: OncoImmunology

Article Title: TNBC-derived Gal3BP/Gal3 complex induces immunosuppression through CD45 receptor

doi: 10.1080/2162402x.2023.2246322

Figure Lengend Snippet: Figure 5. Effect of CD45 blocking on Gal3BP/Gal3 protein-induced immunosuppression. a. Percentage of Treg cells (CD4+/CD25+FOXP3+) in PBMCs treated with EX compared to TEX or Gal3BP/Gal3 complex ± CD45 blocking. Results are expressed as mean±SE (n = 4) (p value by ANOVA). b. Representative dot blot of FACS data showing percentage gated CD4 cells expressing CD25-APC and FOXP3-FITC (Treg cells) in EX- or TEX-treated cells. c. IL-10 and IL-35 were detected in supernatants of PBMCs treated with either TEX or EX from the different experiments. IL-10 secretion by cells. Results are expressed as mean±SE (n = 6) (p value by ANOVA). d. IL-35 secretion by cells. Results are expressed as mean±SE (n = 7); (p values by ANOVA). e. IFNγ secretion of lymphocytes incubated with TEX or TEX+GW4869 (an inhibitor of exosome secretion) and with/without CD45 blocking. f. Percentage of activated T and NK cells after incubation of lymphocytes with EX or TEX with/without CD45 blocking. Results are expressed as mean±SE (n = 4); p values by ANOVA). g. Representative dot blot of FACS data.

Article Snippet: The CD45 inhibitor (CAS 345,630-40-2, 2.9 μM; Calbiochem) is a PTPase CD45 inhibitor and a CD45 blocking peptide.19 Cells were transferred to FACS tubes and stained with antiCD3-PC5.5 (Beckman Coulter), anti-Gal3-PE (R&D Systems), anti-mouse Gal3BP (Novus Biologicals) and mouse antihuman FITC (Jackson ImmunoResearch).

Techniques: Blocking Assay, Dot Blot, Expressing, Incubation

Journal: OncoImmunology

Article Title: TNBC-derived Gal3BP/Gal3 complex induces immunosuppression through CD45 receptor

doi: 10.1080/2162402x.2023.2246322

Figure Lengend Snippet: Figure 6. A new TNBC immunosupressive mechanism. Gal3BP/Gal3 complex secreted by TNBC cells induces immunosuppression through the CD45 receptor on T cells.

Article Snippet: The CD45 inhibitor (CAS 345,630-40-2, 2.9 μM; Calbiochem) is a PTPase CD45 inhibitor and a CD45 blocking peptide.19 Cells were transferred to FACS tubes and stained with antiCD3-PC5.5 (Beckman Coulter), anti-Gal3-PE (R&D Systems), anti-mouse Gal3BP (Novus Biologicals) and mouse antihuman FITC (Jackson ImmunoResearch).

Techniques:

Journal: Frontiers in Psychiatry

Article Title: Galectin-3 Binding Protein, Depression, and Younger Age Were Independently Associated With Alexithymia in Adult Patients With Type 1 Diabetes

doi: 10.3389/fpsyt.2021.672931

Figure Lengend Snippet: Results from biochemical analyses compared between 44 T1D patients with and 248 T1D patients without alexithymia.

Article Snippet: Galectin-3 binding protein (Gal3BP) is a macrophage scavenger receptor which, while activated, induces a number of pro-inflammatory cytokines ( ).

Techniques:

Journal: Frontiers in Psychiatry

Article Title: Galectin-3 Binding Protein, Depression, and Younger Age Were Independently Associated With Alexithymia in Adult Patients With Type 1 Diabetes

doi: 10.3389/fpsyt.2021.672931

Figure Lengend Snippet: Associations with alexithymia in 292 T1D patients explored for three different models.

Article Snippet: Galectin-3 binding protein (Gal3BP) is a macrophage scavenger receptor which, while activated, induces a number of pro-inflammatory cytokines ( ).

Techniques:

Journal: Frontiers in Psychiatry

Article Title: Galectin-3 Binding Protein, Depression, and Younger Age Were Independently Associated With Alexithymia in Adult Patients With Type 1 Diabetes

doi: 10.3389/fpsyt.2021.672931

Figure Lengend Snippet: Associations with high levels of HbA1c.

Article Snippet: Galectin-3 binding protein (Gal3BP) is a macrophage scavenger receptor which, while activated, induces a number of pro-inflammatory cytokines ( ).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Galectin 3–binding protein suppresses amyloid-β production by modulating β-cleavage of amyloid precursor protein

doi: 10.1074/jbc.RA119.008703

Figure Lengend Snippet: Identification of GAL3BP as a factor suppressing Aβ production. a , filtration. Conditioned media from HEK293 cells ( Cont .) and HEK293 cells expressing GPLD1 (+ GPLD1 ) were subjected to Amicon Ultra 30-kDa centrifugal filter units. H4-APPsw cells were incubated with the concentrated and flow-through fractions to test their suppressive activity on the production of Aβ40 and Aβ42. b , lectin affinity beads. The concentrated fractions of a were subjected to lectin affinity beads (WGA-, WFL-, ConA-, ECA-, and VVA-beads), and the unbound fractions were tested for their inhibitory activity on the production of Aβ40 and Aβ42. c , WFL-affinity beads. Materials bound to WFL-beads were eluted with GalNAc, and the eluted fractions were tested for their suppressive activity on the production of Aβ40 and Aβ42. d , ion-exchange columns. The eluted fractions from WFL-beads were subjected to ion-exchange columns. The bound materials were eluted with NaCl (50, 100, 150, 200, 500, and 1000 m m ), and the elution fractions were tested for their suppressive activity on the production of Aβ40. e , silver staining of the purified fractions. Eluted fractions from the ion-exchange column with 0.5 m NaCl were subjected to SDS-PAGE and silver-stained. A band around 90 kDa in the purified fractions ( arrowhead ) was subjected to MS analysis to identify the protein. f , expression of GAL3BP in HEK293 cells. The cDNA encoding GPLD1 was transfected into HEK293 cells. The amounts of GAL3BP proteins in the cell culture media and in the cell lysates and that of GPLD1 in the cell lysates were analyzed by Western blotting. β-Actin was used as a loading control. a–d , the values in the graphs are presented relative to the corresponding controls ( n = 3, mean ± S.D. ( error bars )). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control by one-way ANOVA with Tukey's post-hoc test.

Article Snippet: The following monoclonal or polyclonal antibodies were used: mouse monoclonal anti-GPLD1 antibody (38A1 ab51356-100, Abcam, Cambridge, UK), goat polyclonal anti-GAL3BP antibody (AF2226, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-BACE1 antibody (ab10716, Abcam), rabbit polyclonal anti-ADAM10 antibody (ab1997, Abcam), rabbit polyclonal anti-APP and CTF antibody (A8717, Sigma-Aldrich), mouse monoclonal anti-APP antibody (6E10, Biolegend, San Diego, CA), rabbit polyclonal anti-NRG1 antibody (ab53104 Abcam), rabbit polyclonal antibodies against the carboxyl termini of human sAPPα and sAPPβ , mouse monoclonal anti-c-Myc tag antibody (9E10, Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit polyclonal anti-FLAG tag (DDDDK) antibody (PM020, Medical and Biological Laboratories Co., Ltd. (MBL), Nagoya, Japan), and mouse monoclonal anti-β-actin antibody (A1978, Sigma-Aldrich).

Techniques: Filtration, Expressing, Incubation, Activity Assay, Silver Staining, Purification, SDS Page, Staining, Transfection, Cell Culture, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Galectin 3–binding protein suppresses amyloid-β production by modulating β-cleavage of amyloid precursor protein

doi: 10.1074/jbc.RA119.008703

Figure Lengend Snippet: GAL3BP-induced inhibition of Aβ production. a , the amounts of Aβ40 and Aβ42 in the conditioned media from H4-APPsw cells ( Cont .) and H4-APPsw cells overexpressing GAL3BP (+ GAL3BP ) were measured using ELISAs. b , conditioned media were prepared from control HEK293 cells ( Cont ., lane 1 ), HEK293 cells with GAL3BP knockdown via RNAi (+ RNAi , lane 2 ), and HEK293 cells with GAL3BP knockdown and GPLD1 overexpression (+ RNAi , + GPLD1 , lane 3 ) and subsequently added to H4-APPsw cells to test for their suppressive activity on Aβ production. Western blot analysis confirmed the efficiency of RNAi for the amount of GAL3BP expression in the culture media ( bottom ). c , recombinant GAL3BP (5 μg/ml) prepared from HEK293 cells was added to H4-APPsw cell cultures, and the amounts of Aβ40 and Aβ42 in the media were determined. d , commercially available recombinant GAL3BP was added to H4-APPsw cell culture, and the amounts of Aβ40 and Aβ42 in the media were measured. a–d , the values in this graph are presented relative to the corresponding controls ( n = 3, mean ± S.D. ( error bars )). If error bars representing S.D. values are not visible, they are too small to be displayed. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control by one-way ANOVA with Tukey's post-hoc test.

Article Snippet: The following monoclonal or polyclonal antibodies were used: mouse monoclonal anti-GPLD1 antibody (38A1 ab51356-100, Abcam, Cambridge, UK), goat polyclonal anti-GAL3BP antibody (AF2226, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-BACE1 antibody (ab10716, Abcam), rabbit polyclonal anti-ADAM10 antibody (ab1997, Abcam), rabbit polyclonal anti-APP and CTF antibody (A8717, Sigma-Aldrich), mouse monoclonal anti-APP antibody (6E10, Biolegend, San Diego, CA), rabbit polyclonal anti-NRG1 antibody (ab53104 Abcam), rabbit polyclonal antibodies against the carboxyl termini of human sAPPα and sAPPβ , mouse monoclonal anti-c-Myc tag antibody (9E10, Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit polyclonal anti-FLAG tag (DDDDK) antibody (PM020, Medical and Biological Laboratories Co., Ltd. (MBL), Nagoya, Japan), and mouse monoclonal anti-β-actin antibody (A1978, Sigma-Aldrich).

Techniques: Inhibition, Control, Knockdown, Over Expression, Activity Assay, Western Blot, Expressing, Recombinant, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Galectin 3–binding protein suppresses amyloid-β production by modulating β-cleavage of amyloid precursor protein

doi: 10.1074/jbc.RA119.008703

Figure Lengend Snippet: APP processing by GAL3BP-treatments. H4-APPsw cells were treated with (+ GAL3BP ) or without ( Cont .) the commercially available GAL3BP. a and b , sAPPα and sAPPβ proteins in the culture media and CTFs in the cell lysates were detected with Western blot analysis. Blots with β-actin were used as a loading control. The relative sAPPβ and sAPPα in the culture media from GAL3BP-treated cells were normalized to the control samples. The relative CTFs were normalized to the corresponding β-actin levels. c , the amounts of sAPPβ and sAPPα in the conditioned media and βCTF in the cell lysates were measured using ELISAs. d , the amounts of Aβ40 and Aβ42 in the lysate were measured using ELISAs. e , Western blot analysis of BACE1 and ADAM10 in the lysates of H4-APPsw cells treated with or without GAL3BP. Blots with β-actin were used as a loading control. The relative BACE1 and ADAM10 values are normalized to the corresponding β-actin levels. p > 0.05 versus control by one-way ANOVA with Tukey's post-hoc test. b–e , the controls were vehicle controls, and the values in this graph are presented relative to each control ( n = 3, mean ± S.D. ( error bars )). *, p < 0.05; ***, p < 0.001 versus control by one-way ANOVA with Tukey's post-hoc test.

Article Snippet: The following monoclonal or polyclonal antibodies were used: mouse monoclonal anti-GPLD1 antibody (38A1 ab51356-100, Abcam, Cambridge, UK), goat polyclonal anti-GAL3BP antibody (AF2226, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-BACE1 antibody (ab10716, Abcam), rabbit polyclonal anti-ADAM10 antibody (ab1997, Abcam), rabbit polyclonal anti-APP and CTF antibody (A8717, Sigma-Aldrich), mouse monoclonal anti-APP antibody (6E10, Biolegend, San Diego, CA), rabbit polyclonal anti-NRG1 antibody (ab53104 Abcam), rabbit polyclonal antibodies against the carboxyl termini of human sAPPα and sAPPβ , mouse monoclonal anti-c-Myc tag antibody (9E10, Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit polyclonal anti-FLAG tag (DDDDK) antibody (PM020, Medical and Biological Laboratories Co., Ltd. (MBL), Nagoya, Japan), and mouse monoclonal anti-β-actin antibody (A1978, Sigma-Aldrich).

Techniques: Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Galectin 3–binding protein suppresses amyloid-β production by modulating β-cleavage of amyloid precursor protein

doi: 10.1074/jbc.RA119.008703

Figure Lengend Snippet: Interaction of APP and GAL3BP. a–d , inhibitory effects of GAL3BP on the processing of immunoprecipitated APP by BACE1. Immunoprecipitated APP from H4-APPsw cells were treated with BACE1 (2.5 unit/ml) in the presence or absence of the commercially available GAL3BP (10 μg/ml) ( a and b ). Immunoprecipitated APP was also treated with BACE1 (2.5 unit/ml) and BACE1 inhibitor (50 n m ) ( c and d ). The samples were then analyzed with Western blotting ( a and c ) and ELISAs to detect full-length ( FL ) APP and βCTF. The relative values for full-length APP and βCTF were normalized ( b and d ). The whole gel images are shown in Fig. S13 . The values in this graph are presented relative to each other and the corresponding controls ( n = 3, mean ± S.D. ( error bars )). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control by one-way ANOVA with Tukey's post-hoc test. e , direct interaction of APP and GAL3BP. The immunoprecipitated APP proteins from H4-APPwt cells were incubated with the commercially available GAL3BP, and then the bound materials were analyzed with Western blotting using antibodies against APP and GAL3BP. Nonimmunoglobulin was used as a control. f , co-immunoprecipitation of GAL3BP and APP. The cDNA encoding GAL3BP with the c-Myc tag was transfected into H4-APPsw cells. The cell lysates were subjected to immunoprecipitation with anti-APP or anti-c-Myc antibodies, and then immunoprecipitated materials were analyzed with Western blotting. The blots were probed with anti-APP ( top panels ) and anti-GAL3BP ( bottom panels ) antibodies. Nonimmunoglobulin was used as a control. g and h , effect of GAL3BP on NRG1 processing. H4-APPsw cells were treated with the commercially available GAL3BP or the BACE1 inhibitor, and the cell lysates were analyzed with Western blotting using the antibody to NRG1. Blots with β-actin were used as a loading control. The values in this graph are presented relative to each other and the corresponding controls ( n = 3, mean ± S.D.). *, p < 0.05 versus control by one-way ANOVA with Tukey's post-hoc test.

Article Snippet: The following monoclonal or polyclonal antibodies were used: mouse monoclonal anti-GPLD1 antibody (38A1 ab51356-100, Abcam, Cambridge, UK), goat polyclonal anti-GAL3BP antibody (AF2226, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-BACE1 antibody (ab10716, Abcam), rabbit polyclonal anti-ADAM10 antibody (ab1997, Abcam), rabbit polyclonal anti-APP and CTF antibody (A8717, Sigma-Aldrich), mouse monoclonal anti-APP antibody (6E10, Biolegend, San Diego, CA), rabbit polyclonal anti-NRG1 antibody (ab53104 Abcam), rabbit polyclonal antibodies against the carboxyl termini of human sAPPα and sAPPβ , mouse monoclonal anti-c-Myc tag antibody (9E10, Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit polyclonal anti-FLAG tag (DDDDK) antibody (PM020, Medical and Biological Laboratories Co., Ltd. (MBL), Nagoya, Japan), and mouse monoclonal anti-β-actin antibody (A1978, Sigma-Aldrich).

Techniques: Immunoprecipitation, Western Blot, Control, Incubation, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Galectin 3–binding protein suppresses amyloid-β production by modulating β-cleavage of amyloid precursor protein

doi: 10.1074/jbc.RA119.008703

Figure Lengend Snippet: Immunofluorescence analysis of the intracellular location of extracellularly added GAL3BP. a , H4-APPsw cells were incubated with extracellularly applied recombinant GAL3BP (5 μg/ml) for 24 h and then stained with anti-GAL3BP (c-Myc) antibody. Arrowheads , intracellular signals of recombinant GAL3BP. Vehicle treatment was used as negative control ( N.C. ). Scale bars , 50 μm. b , H4-APPsw cells treated with GAL3BP were co-stained for GAL3BP (c-Myc), the early endosome marker EEA1, and nuclei ( DAPI ). Arrowheads , co-localization of intracellular signals of recombinant GAL3BP and EEA1. Scale bars , 20 μm. c , H4-APPsw cells were pre-incubated with or without nystatin (50 μg/ml) for 1 h and then incubated with recombinant GAL3BP for 3 h. The cells were stained for GAL3BP ( red ) and DAPI ( blue ). Asterisks , cells with no signal for intracellular GAL3BP. Arrowheads , intracellular dotlike signals of recombinant GAL3BP. Arrows , large grainlike signals for GAL3BP. Scale bars , 20 μm. d , quantification of intracellular signals for GAL3BP. Cells were classified into three groups: no intracellular signals, intracellular dotlike signals, and large grainlike signals (corresponding to asterisks , arrowheads , and arrows in c , respectively). More than 500 cells were counted in randomly taken fields and statistically analyzed using the χ 2 test. e and f , H4-APPsw cells were treated with the recombinant GAL3BP (c-Myc–tagged). The cells were co-stained for APP, EEA1, and nuclei (DAPI) ( e ). Arrowheads , co-localization of intracellular signals of APP and EEA1 ( e ). The cells were also subjected to a PLA using antibodies to APP and c-Myc ( f ). Red signals indicate the co-localization of intracellular signals of APP and recombinant GAL3BP ( f ). Scale bars , 20 μm.

Article Snippet: The following monoclonal or polyclonal antibodies were used: mouse monoclonal anti-GPLD1 antibody (38A1 ab51356-100, Abcam, Cambridge, UK), goat polyclonal anti-GAL3BP antibody (AF2226, R&D Systems, Minneapolis, MN), rabbit polyclonal anti-BACE1 antibody (ab10716, Abcam), rabbit polyclonal anti-ADAM10 antibody (ab1997, Abcam), rabbit polyclonal anti-APP and CTF antibody (A8717, Sigma-Aldrich), mouse monoclonal anti-APP antibody (6E10, Biolegend, San Diego, CA), rabbit polyclonal anti-NRG1 antibody (ab53104 Abcam), rabbit polyclonal antibodies against the carboxyl termini of human sAPPα and sAPPβ , mouse monoclonal anti-c-Myc tag antibody (9E10, Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit polyclonal anti-FLAG tag (DDDDK) antibody (PM020, Medical and Biological Laboratories Co., Ltd. (MBL), Nagoya, Japan), and mouse monoclonal anti-β-actin antibody (A1978, Sigma-Aldrich).

Techniques: Immunofluorescence, Incubation, Recombinant, Staining, Negative Control, Marker