mouse anti bag1  (Santa Cruz Biotechnology)

Journal: Cell Stress & Chaperones

Article Title: Bag1 protein loss sensitizes mouse embryonic fibroblasts to glutathione depletion

doi: 10.1016/j.cstres.2024.05.003

Figure Lengend Snippet: Generation of Bag1 exon 5 mutant mice. (a) Structure of human BAG1 and mouse Bag1 genes and the coding regions of Bag1L. Lines and boxes indicate the Bag1 gene and seven exons (Ex1–Ex7), respectively. Small arrows indicate primers for PCR to verify the genotypes of mice. Bag1 protein was illustrated with the UbL and BAG domains (shadowed boxes). Dark shadowed boxes indicate the BAG domain helix 2, which is encoded in exon 5. The target position of Bag1 is Cys282 (Cys272 in humans). The structures of putative ORFs of mutant Bag1S with the insertion of 49 bp in exon 5 (if generated by normal splicing) and the mutant Bag1S with the deletion of exon 5 due to in-frame exon skipping (see below) are shown in a square box. (b) Genotyping of F1 pups from the candidate Bag1 mutant mouse. A genome-edited F0 candidate was mated with a Bag1 WT C57/BL6J mouse to generate F1 pups. To distinguish the Bag1 mutant mice, the exon 5 region was amplified from 8 F1 pups by PCR and analyzed by agarose gel electrophoresis. Arrows indicate the PCR products of Bag1 WT (WT) and mutated exon 5. Pups with the mutant allele are shown using boldface numbers. (c) Genotyping of mice F2 pups from the possible F1 mice carrying the hetero allele (#1 mated with #6). The genome was analyzed by PCR. (d) Bag1 mutant genome sequence is shown in alignment with the Bag1 WT sequence. A homozygous mouse (#29) genome was amplified by PCR. The exon 5 region is indicated. The full and partial oligo DNA sequences in the #29 genome are underlined. Abbreviations used: ORF, open reading frame; UbL, ubiquitin-like; PCR, polymerase chain reaction; WT, wild type.

Article Snippet: Immunoblotting was performed using antibodies against Bag1 (AF815, R&D systems, Minneapolis, MN, USA), Gsr (sc-133245, Santa Cruz Biotechnology, Dallas, TX, USA), and Glyceraldehyde 3-phosphate dehydrogenase (G8795, Merk, Darmstadt, Germany), respectively.

Techniques: Mutagenesis, Generated, Amplification, Agarose Gel Electrophoresis, Sequencing, Ubiquitin Proteomics, Polymerase Chain Reaction

Journal: Cell Stress & Chaperones

Article Title: Bag1 protein loss sensitizes mouse embryonic fibroblasts to glutathione depletion

doi: 10.1016/j.cstres.2024.05.003

Figure Lengend Snippet: The mutant exon 5 is skipped by splicing to generate alternative mRNA encoding mutant Bag1 with helix 3 deletion that is undetectable. (a) Analysis of Bag1 cDNA. RT-PCR of Bag1 mRNA using the indicated primer pair generated 1281 bp of cDNA product of Bag1 WT (WT). The exon 5 mutant exhibited an approximately 100-bp smaller cDNA size. Pups carrying only the shorter cDNA (exon 5 mutant Bag1 ) are shown in boldface numbers. (b) Genes and mRNA structures of Bag1 WT and Bag1 Δex5 mice are illustrated with the frequency of pups' appearance. The inserted oligo on exon 5 in a mutant mouse is indicated by pink coloration. Arrowheads indicate primers to verify the genotypes of mice and clone cDNA. The frequency of live birth among Bag1 Δex5 mice are shown. (c) Homo Bag1 WT and Bag1 Δex5 mouse embryos at E13.5 are shown. (d) Expression of Bag1 mRNA in male Bag1 WT/WT and Bag1 Δex5/Δex5 mice was examined using RT-PCR. Total RNAs from organs and tissues derived from ectoderm, mesoderm, or endoderm are shown. Positions of Bag1 WT (WT) and Bag1 Δex5 (Δ5) PCR fragments are indicated by arrows. (e) Bag1 proteins in male Bag1 WT (WT) and Bag1 Δex5 (Δ5) mice were examined. Positions of Bag1L, Bag1S, and GAPDH are indicated. Protein lysate of Bag1 WT MEFs (M) was used as positive controls. The 63 kDa protein that appeared in both WT and Δ5 lysates is a non-Bag1 protein that reacted with the monoclonal antibody used, as it could not be detected with the anti-Bag1 rabbit antibody (see ). Abbreviations used: MEF, mouse embryonic fibroblast; RT-PCR, reverse transcription-PCR; cDNA, complementary DNA; mRNA, messenger ribonucleic acid; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; WT, wild type; PCR, polymerase chain reaction.

Article Snippet: Immunoblotting was performed using antibodies against Bag1 (AF815, R&D systems, Minneapolis, MN, USA), Gsr (sc-133245, Santa Cruz Biotechnology, Dallas, TX, USA), and Glyceraldehyde 3-phosphate dehydrogenase (G8795, Merk, Darmstadt, Germany), respectively.

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Generated, Expressing, Derivative Assay, Reverse Transcription, Polymerase Chain Reaction

Journal: Cell Stress & Chaperones

Article Title: Bag1 protein loss sensitizes mouse embryonic fibroblasts to glutathione depletion

doi: 10.1016/j.cstres.2024.05.003

Figure Lengend Snippet: Effect of the insertion of a neo marker knockout cassette upstream of Bag1 on head-to-head gene Chmp5 expression. (a) Chromosomal region of Bag1 (exon 1–7)-Chmp5 (exon 1–8), including the predicted regulatory future from the Ensemble regulation resources ( https://oct2022.archive.ensembl.org/Mus_musculus/Gene/Summary?db=core;g=ENSMUSG00000028419;r = 4:40945802–40951599;t = ENSMUST00000030128;gene_summary=regulatory_build=normal ). Constructs of Chmp5-fused nLuc reporter genes carrying 8.4 kbp (F1-cLuc) and 5.0 kbp (F3-cLuc) are shown. The upstream region was replaced with the reporters, with the neo cassettes illustrated (F1-neo-nLuc and F3-neo-nLuc; see text for detail). (b) nLuc activity was observed by 3 different experiments (n = 3). Normalized activity of F1-nLuc (F1), F1-neo-nLuc (F1neo), F3-nLuc (F3), and F3-neo-nLuc (F3neo) is shown. The values are shown as the means ± SEM. Significance was determined using the Student’s t-test. Abbreviation used: SEM, standard error of the mean.

Article Snippet: Immunoblotting was performed using antibodies against Bag1 (AF815, R&D systems, Minneapolis, MN, USA), Gsr (sc-133245, Santa Cruz Biotechnology, Dallas, TX, USA), and Glyceraldehyde 3-phosphate dehydrogenase (G8795, Merk, Darmstadt, Germany), respectively.

Techniques: Marker, Knock-Out, Expressing, Construct, Activity Assay

Journal: Cell Stress & Chaperones

Article Title: Bag1 protein loss sensitizes mouse embryonic fibroblasts to glutathione depletion

doi: 10.1016/j.cstres.2024.05.003

Figure Lengend Snippet: Establishment and characterization of Bag1 Δex5 MEFs. (a) Bag1 protein expression in Bag1 WT and Bag1 Δex5 MEFs was examined by immunoblotting. Bag1 isoforms (Bag1L and Bag1S) are indicated by arrows. GAPDH signals were used as a loading control. No stress (Cont, lanes 1 and 3), and 100 μM BSO for 30 min (BSO, lanes 2 and 4) groups are shown. (b) Relative sensitivity of Bag1 Δex5 MEF to that of Bag1 WT MEF, and the indicated concentration of DOX for 24 h. Relative growth levels were normalized against 0 µM values set as 100%, with the values shown as means ± SEM (n = 3, i.e., 3 independent experiments). (c) Synthesis and redox cycles of GSH. Glutamate cysteine ligase (GCL) is responsible for the first step of GSH synthesis. GSH is regenerated by GSSG reduction by glutathione reductase (GR). GR and glutathione peroxidase (GPx) are also indicated. BSO inhibited GCL. (d) Sensitivity to BSO was determined over 24 h. Growth levels (%) were shown as the means ± SEM from three independent experiments. * P < 0.05 and ** P < 0.01 when compared to viability without BSO (one-way analysis of variance followed by a Dunnett’s post hoc test for multiple parameter comparisons). (e) Intracellular GSH/GSSG ratios in Bag1 WT and Bag1 Δex5 MEFs were determined with and without BSO (200 μM BSO for 24 h). The values are shown as the means ± SEM from four independent experiments. The P -values are indicated using Student's t -test. (f) GR protein levels in Bag1 WT and Bag1 Δex5 MEFs were determined by immunoblotting using an anti-GR antibody. β-tubulin signals were used as a loading control. MEFs were treated with BSO (100 μM for 24 h, lanes 2 and 4). No treatment controls are shown (lanes 1 and 3). (g) Cellular H 2 O 2 levels were determined for Bag1 WT and Bag1 Δex5 MEFs. The values are shown as the means ± SEM from four independent experiments. The P -values are indicated using Student's t-test. Abbreviations used: BSO, buthionine sulfoximine; DOX, doxorubicin; GSH, glutathione; GSSG, oxidized glutathione; MEFs, mouse embryonic fibroblasts; SEM, standard error of the mean; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Immunoblotting was performed using antibodies against Bag1 (AF815, R&D systems, Minneapolis, MN, USA), Gsr (sc-133245, Santa Cruz Biotechnology, Dallas, TX, USA), and Glyceraldehyde 3-phosphate dehydrogenase (G8795, Merk, Darmstadt, Germany), respectively.

Techniques: Expressing, Western Blot, Control, Concentration Assay

Journal: Journal of Cancer

Article Title: ALDH3A1 driving tumor metastasis is mediated by p53/BAG1 in lung adenocarcinoma

doi: 10.7150/jca.58250

Figure Lengend Snippet: ALDH3A1 regulates tumor cell proliferation through p53/BAG1 axis. The correlation between ALDH3A1 and BAG1 (A) or TP53 (B) in GEPIA. (C) BAG1 expression in NC, ALDH3A1-sh1 and ALDH3A1-sh2 cell lines. (D) The expression of BAG1, p53 and β-actin detected by western blot in NC, ALDH3A1-sh1 and ALDH3A1-sh2 cell lines. (E) ALDH3A1 and BAG1 expression in tumor cell lines treated with p53 inhibitor. (F) Tumor cell growth from A549 cell line transduced with BAG1-sh by CCK-8, and absorbance per well was measured at 450 nm using an automatic microplate reader. (G) MFI of Ki67 expression detected by flow cytometry in tumor cell transduced with BAG1-sh. (H) Representative images of invasion and statistical analysis of invasion cell numbers per field in A549 cell line transduced with BAG1-sh. Data were expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Next, the membranes were blockaded by 10% skim milk powder, followed by incubation with the following primary antibodies overnight at 4 °C: β-actin (8H10D10) Mouse mAb #3700 (CST, USA), p53 (DO-7) Mouse mAb #48818 (CST, USA), Bag1 (3.10G3E2) Mouse mAb #3920 (CST, USA), and anti-ALDH3A1 antibody (ab186726) (Abcam, UK).

Techniques: Expressing, Western Blot, Transduction, CCK-8 Assay, Flow Cytometry

Journal: Journal of Cancer

Article Title: ALDH3A1 driving tumor metastasis is mediated by p53/BAG1 in lung adenocarcinoma

doi: 10.7150/jca.58250

Figure Lengend Snippet: Low expression of BAG1 is associated with good prognosis. (A) Correlation analysis of BAG1 and ALDH3A1 in enrolled LUAD patients. Correlation analysis between BAG1 and cancer stem cell-associated genes (B) or EMT-associated genes (C). (D) BAG1 expression in M0 and M1 LUAD patients. (E) IHC staining for ALDH3A1 and BAG1 in LUAD cancer tissues and (F) correlation analysis between BAG1 and ALDH3A1. (G) Kaplan-Meier survival curve of enrolled LUAD patients with high or low expression of BAG1 within tumor specimens. X-axis represents the survival time, and Y-axis represents the survival ratio of patients. Data were expressed as mean ± SEM. * p < 0.05, ** p < 0.01.

Article Snippet: Next, the membranes were blockaded by 10% skim milk powder, followed by incubation with the following primary antibodies overnight at 4 °C: β-actin (8H10D10) Mouse mAb #3700 (CST, USA), p53 (DO-7) Mouse mAb #48818 (CST, USA), Bag1 (3.10G3E2) Mouse mAb #3920 (CST, USA), and anti-ALDH3A1 antibody (ab186726) (Abcam, UK).

Techniques: Expressing, Immunohistochemistry

Journal: The American Journal of Pathology

Article Title: Penetration of CD8 + Cytotoxic T Cells into Large Target, Tissue Cysts of Toxoplasma gondii , Leads to Its Elimination

doi: 10.1016/j.ajpath.2019.04.018

Figure Lengend Snippet: CD8+ immune T cells eliminate pre-existing cysts of Toxoplasma gondii through perforin-dependent cytotoxic activity. Severe combined immunodeficiency (SCID) mice were infected orally with 10 cysts of the ME49 strain of T. gondii and treated with sulfadiazine beginning at 10 days after infection to establish a chronic infection by forming cysts in their brains. A and B: CD8+ immune T cells (2.1 × 106 cells) purified from the spleens of chronically infected wild-type (WT) or Prf1−/− mice were injected intravenously from a tail vein, and 7 days later, amounts of mRNA for bradyzoite (cyst)-specific BAG1 and CST1 and tachyzoite-specific SAG1 (A) and CD3, a T-cell surface marker (B), were measured by real-time RT-PCR in the brains of the recipient animals. C: Amounts of mRNA for interferon (IFN)-γ and effector molecules [inducible nitric oxide synthase 2 (NOS2), immunity-related GTPases M3 (Irgm3), and guanylate-binding protein 1 (Gbp1)] of the IFN-γ–mediated protective immunity to prevent tachyzoite proliferation were also measured by real-time RT-PCR. P values were obtained using t-test, and corrected P values shown in the figure were calculated by multiplying the P values by the number of comparisons performed among three groups. D and E: Immunohistochemical detection of T cells in the parenchyma (D) and a perivascular area (E) of the brains of infected nude mice at 2 to 3 days after a systemic transfer of Prf1−/− CD8+ immune T cells (4.2 × 106 cells). The T cells (positive for CD3) were stained in red. Arrows indicate the representatives of the T cells. Data are expressed as means ± SEM in each group (A–C). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Original magnification, ×200 (D and E).

Article Snippet: 20 Staining for bradyzoite-specific BAG1 and CD3 was performed in the same manner, with a modification of the use of mouse anti-BAG1 monoclonal antibody after blocking with F(ab) 2 fragments of goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) overnight.

Techniques: Activity Assay, Infection, Purification, Injection, Marker, Quantitative RT-PCR, Binding Assay, Immunohistochemical staining, Staining

Journal: The American Journal of Pathology

Article Title: Penetration of CD8 + Cytotoxic T Cells into Large Target, Tissue Cysts of Toxoplasma gondii , Leads to Its Elimination

doi: 10.1016/j.ajpath.2019.04.018

Figure Lengend Snippet: Invasion of T cells into Toxoplasma gondii cysts in the brains of infected mice. A–J: CBA/J mice were infected orally with 10 cysts of the ME49 strain of T. gondii; and 2 months later, their brains were applied for immunohistologic staining for T. gondii (A, B, D, E, G, and I) or bradyzoite-specific BAG1 (C, F, H, and J) in brown in combination with the staining for CD3, the T-cell marker, in red. The entire fields of four or five sagittal sections from each of the brains of four mice were analyzed. A–C: T cells that attached on the surface of cyst-containing cells. A: An arrow indicates the cyst wall. B: The arrow indicates the T cells in a spindle shape. C: The arrow indicates the bent of cyst wall inward at the site of T-cell attachment, and the arrowhead indicates a projection of the cyst wall outward nearby the site of the T-cell attachment. D–F: T cells located halfway through the cyst wall (arrows). G and H: T cells completely penetrated into the cysts were detected (arrows). I and J: Totally destroyed cysts associated with an accumulation of inflammatory cells, including T cells. K: Comparison of the diameters of cysts with and without T-cell association. Original magnification: ×400 (A–H); ×200 (I and J).

Article Snippet: 20 Staining for bradyzoite-specific BAG1 and CD3 was performed in the same manner, with a modification of the use of mouse anti-BAG1 monoclonal antibody after blocking with F(ab) 2 fragments of goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) overnight.

Techniques: Infection, Staining, Marker, Cell Attachment Assay, Comparison

Journal: The American Journal of Pathology

Article Title: Penetration of CD8 + Cytotoxic T Cells into Large Target, Tissue Cysts of Toxoplasma gondii , Leads to Its Elimination

doi: 10.1016/j.ajpath.2019.04.018

Figure Lengend Snippet: CD8+ immune T cells attach to and invade into Toxoplasma gondii cysts in the brains of infected mice. Athymic nude mice were infected orally with 20 cysts of the ME49 strain of T. gondii and treated with sulfadiazine beginning at 11 days after infection to establish a chronic infection by forming cysts in their brains. A–F: CD8+ T cells (3.5 × 106 cells) purified from the spleens of infected BALB/c mice were injected intravenously from a tail vein; and 2 to 3 days later, their brains were applied for immunohistochemical staining for T. gondii (brown; A, C, E, and F) or bradyzoite-specific BAG1 (brown; B and D) and CD3 (red), the T-cell marker. A and B: T cells attached on the surface of cyst-containing cells. C–F: T cells that had migrated halfway through the cyst wall (arrows). F: T cells that completely penetrated into the cysts were detected (arrowheads). G–K: Confocal microscopy with staining for T. gondii (green) and CD3 (red) was also performed on their brains. G: The bent of the cyst wall inward at the site of CD8+ T-cell attachment (arrow), shown at higher magnification in H. H: Leakage of T. gondii materials at the site of CD8+ T-cell attachment (arrows). I–K: Confocal images of the T cell that was halfway in the invasion into a cyst. Another group of infected and sulfadiazine-treated nude mice received CD8+ normal T cells from uninfected BALB/c mice, and the immunohistochemical studies were performed on their brains in the same manner. L: The frequencies of T. gondii cysts associated with the T cells were calculated for each of these two groups of mice that had received the normal or immune CD8+ T cells. Data are expressed as means ± SEM in each group (L). ∗P < 0.05 (U-test). Original magnification: ×400 (A–F); ×1000 (G–K). SCID, severe combined immunodeficiency; WT, wild type.

Article Snippet: 20 Staining for bradyzoite-specific BAG1 and CD3 was performed in the same manner, with a modification of the use of mouse anti-BAG1 monoclonal antibody after blocking with F(ab) 2 fragments of goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) overnight.

Techniques: Infection, Purification, Injection, Immunohistochemical staining, Staining, Marker, Confocal Microscopy, Cell Attachment Assay