MA5-14003

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Bax (green) showing staining in the cytoplasm of Hela cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Bax monoclonal antibody (Product # MA5-14003) in 3% BSA-PBS at a dilution of 1:50 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Bax (green) showing staining in the cytoplasm of MCF-7 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Bax monoclonal antibody (Product # MA5-14003) in 3% BSA-PBS at a dilution of 1:50 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Bax (green) showing staining in the cytoplasm of L929 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Bax monoclonal antibody (Product # MA5-14003) in 3% BSA-PBS at a dilution of 1:50 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of Bax in Hela cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde, washed with PBS, and incubated with Bax monoclonal antibody (Product # MA5-14003) at a dilution of 0.5 µg/test for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of Bax in L929 cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde, washed with PBS, and incubated with Bax monoclonal antibody (Product # MA5-14003) at a dilution of 0.5 µg/test for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of Bax in Ramos cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde, washed with PBS, and incubated with Bax monoclonal antibody (Product # MA5-14003) at a dilution of 1 µg/test for 60 min at room temperature. Cells were then blocked in a solution of 2% BSA-PBS for 30 min at room temperature, incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody, and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 Photomicrographs of sections in the cerebral cortex of adult male albino rats. (A) control group showing weak positive immunoreaction for Bax (arrow). (B) diabetic group showing strong positive immunoreaction for Bax (arrows). (C) diabetic cerebral ischemic-reperfused group showing strong positive immunoreaction for Bax (arrows). (D) diabetic group treated with resveratrol showing moderate immunoreaction for Bax (arrows). (E) diabetic cerebral ischemic-reperfused group treated with resveratrol showing moderate immunoreaction for Bax (arrows). [A, B, C, D, E using Avidin-biotin peroxidase stain with Hx counter stain x400 (scale bars represent 10 mum)].

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. Pretreatment with DZX modulates the protein expression of Bcl-2, caspase-12, caspase-3, Bax, CHOP and GRP78 in rats subjected to I/R. Western blot analysis was used to detect the protein expression levels of Bcl-2, caspase-12, caspase-3, Bax, CHOP and GRP78 in hippocampal cells isolated from I/R-treated rats. Rats were treated with DZX with or without 5-HD and subjected to cerebral ischemia for 2 h followed by 12 h of reperfusion. Sham rats were not subjected to cerebral ischemia. DZX, diazoxide; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein; CHOP, C/EBP homologous protein; GRP78, 78 kDa glucose-regulated protein; I/R, ischemia/reperfusion; 5-HD, 5-hydroxydecanoate.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. TPG regulates apoptosis-associated factors in H9C2 cells. H9C2 cells were treated with TPG (10, 40 and 160 ug/ml) for 24 h and subjected to I/R injury. (A) Relative mRNA expression levels of caspase-3, PARP1, Bax and Bcl-2 were investigated by reverse transcription-quantitative polymerase chain reaction. (B) Relative protein expression levels of caspase-3, PARP1, Bax and Bcl-2 were evaluated by western blotting and normalized to beta-actin expression. (C) Blot results were semi-quantified. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 4 Effect of DAS (50, 100, and 200 mg/kg) on Bax immunohistochemical expression in CCL 4 -intoxicated rats. Hepatic sections (scale bar = 50) obtained from (A) control group, (B) CCl 4 group, (C) CCl 4 + DAS50 group, (D) CCl 4 + DAS100 group and (E) CCl 4 +DAS200 group. (F) Quantification of Bax immune-positive cells to the total area of microscopic field across six fields. Values are expressed as mean +- SD of six rats. a P < 0.05 compared with control group; b P < 0.05 compared with CCl 4 group; c P < 0.05 compared with corresponding CCl 4 +DAS50; d P < 0.05 compared with corresponding CCl 4 +DAS100 group. CCl4, carbon tetrachloride; DAS, diallyl sulfide; SD, standard deviation

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 6 The expression of CHCHD2, the dimerization of BAX, and the interaction between BCL-XL and BAX in the aggregates of UEC-derived hiPSCs and WA09 hESCs that were cultured in LDN193189/A83-01-containing and SB431542/IWR-1-containing media. (A) The expression and activation of CHCHD2, BAX, BCL-XL, and CASP3 were revealed by western blotting in the indicated cell aggregate samples that were cultured in the indicated media. (B) Dimerized BAX (~40 kDa) with electromobility shift from BAX monomer (~20 kDa) was detected by western blotting in the cell aggregate samples. (C) BCL-XL interacted and co-immunoprecipitated with BAX in the cell aggregate samples cultured in the SB431542/IWR-1-containing medium was detected by western blotting. 501: UEC715i-501 hiPSCs. 009: UEC001i-009 hiPSCs. L/A: LDN193189/A83-01-containing medium. S/I: SB431542/IWR-1-containing medium. All the cell aggregates were cultured in the indicated media for 6 days prior to sample collection and analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 Proliferation and apoptosis. a Immunohistochemical staining for proliferation marker Ki67 in kidney tissue (left), alongside quantification of the percentage of Ki67-positive cells (right). b Western blot on kidney tissue measuring proliferation markers PCNA, VEGF, survivin, and beta-actin (left) alongside quantification (right). c Immunohistochemical staining for apoptosis marker Caspase-3 in kidney tissue. d Western blot on kidney tissue measuring apoptosis markers BAX, Bcl-2, and Caspase-3 (left) alongside quantification (right). Each group has n = 3 mice. Significant difference a p < 0.05: relative to untreated control; b p < 0.05: relative to AKI; c p < 0.05: relative to AKI-EV; d p < 0.05: relative to AKI-EV-pFUS

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 ( a , b ) Protein expression and molecular mechanisms of EA inhibitory actions in SKBR3 cell line. This plant extract induces an overexpression of E-cadherin, beta-catenin, and downregulation of vimentin and fascin, while upregulating pro-apoptotic markers (Bax and Caspase-3), in comparison with their control and inhibiting anti-apoptotic markers (Bcl-2). Furthermore, EA plant extract inhibits the phosphorylation of ErbB2 and beta-catenin, as well as the expression of JNK1/2/3. beta-actin was used as a control for the proteins amount in this assay. Cells were treated with 100 and 200 muL/mL of EA extract for 48 h, as explained in the materials and methods and the results sections. ( a ) Blot image and ( b ) quantification of bands.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 8 ( a , b ) Protein expression and molecular mechanisms of EA inhibitory actions in ZR75 cell line. This plant extract induces an overexpression of E-cadherin, beta-catenin, and downregulation of vimentin and fascin; in addition, pro-apoptotic markers Bax and Caspase-3 are upregulated in comparison with their control, while anti-apoptotic marker Bcl-2 is inhibited. Furthermore, EA plant extract inhibits the phosphorylation of ErbB2 and beta-catenin, as well as JNK1/2/3 expression. beta-actin served as a control in this assay. Cells were treated with 100 and 200 muL/mL of EA extract for 48 h, as explained in the materials and Methods section. ( a ) Blot image and ( b ) quantification of bands.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 MiR-203-3p and Sema3A regulated the apoptosis of HG-induced podocytes. HG-induced podocytes were transfected with miR-con, miR-203-3p, miR-203-3p + pcDNA or miR-203-3p + Sema3A. (a) Podocyte apoptosis was evaluated using flow cytometry. (b) The activity of caspase-3 was measured by the Caspase-3 Activity Detection Kit. (c and d) The WB analysis determined the protein levels of apoptosis-associated markers Bax and Bcl-2. * P < 0.05.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 3 Desoxyrhapontigenin ameliorates neuronal apoptosis in the brain tissues of rats with anaesthesia-induced neuronal injury. a TUNEL-positive cells; b Protein expression of caspase-2, Bcl-2, and Bax by Western blotting. Mean +- SEM (n = 15); ## p < 0.01 vs. controls; ** p < 0.01 vs. ISF group

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Flow cytometry analysis of negative control cells acquired without stain for Bcl2, Bax and Caspase 3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 ( C ) Flow cytometry analysis of cell counts, detecting the number of Bcl-2 positive cells. ( D ) Flow cytometry analysis of cell counts, detecting the number of Bax positive cells. ( E ) Flow cytometry analysis of cell counts, detecting the number of Caspase 3 positive cells. 1: GMSCs control; 2: GMSCs treated with Schiff base complex; 3: GMSCs treated with cisplatin; 4: GMSCs treated with Schiff base complex and cisplatin together; 5: SCC cells; 6: SCC treated with Schiff base complex; 7: SCC treated with cisplatin; 8: SCC treated with Schiff base complex and cisplatin together.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Overexpression of miR-509-3p inhibited stemness, cisplatin resistance and cell growth, and promoted apoptosis of A549-dereived stem cells. A qRT-PCR analysis on miR-509-3pin spheres of A549-derived stem cells, * p < 0.01 compared with parental group. B qRT-PCR on the expression of stemness-associated gens (SOX2, CD44, and CD133) in A549cell spheres 48 h after transfection with mimic miRNAs, * p < 0.01 compared with Mock. C , D Number of tumor spheres in miR-509-3p overexpressing A459 stem cells, * p < 0.01 compared with Mock. E Flow cytometry analysis of cell cycle in A549-dereived stem cells 48 h after overexpression of miR-509-3p, * p < 0.01 compared with Mock. F qRT-PCR analysis of miR-509-3pin A549 and cisplatin-resistant A549/R cells. * p < 0.01 compared with A549. G CCK-8 assay on the cell viability of miR-509-3p-overexpressing A549-dereived stem cells treated with cisplatin at various concentration (0 muM, 4 muM, 8 muM, 16 muM, 32 muM), * p < 0.01 compared with Mock. H , I Annexin V-FITC/PI staining of A549-dereived stem cells 48 h after miR-509-3p overexpression, * p < 0.01 compared with Mock. J Western blotting analysis on expression of apoptosis-associated genes (Bax, Bcl-2 and cleaved-caspase-3) 48 h after transfection with miR-509-3p or Mock, * p < 0.01 compared with Mock. K , L Colony formation assay on cell growth of A549-dereived stem cells after 15 days culture, * p < 0.01 compared with Mock. M qRT-PCR analysis on proliferation-associated genes (Cyclin

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 Overexpression of ASAP1-IT1 blocked miR-509-3p-mediated cell functions in cancer stem cells both in vitro and in vivo . A Number of A549 cell tumor spheres after expression of Mock, pcDNA, miR-509-3p, and ASAP1-IT1, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01 compared with miR-509-3p + pcDNA, F p < 0.01 compared with miR-509-3p + ASAP1-IT1. B qRT-PCR analysis on stemness-related genes (SOX2, CD44, and CD133) in A549 stem cells, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01compared with miR-509-3p + pcDNA. C Annexin V-FITC/PI staining of A549 stem cells with Mock, pcDNA, miR-509-3p, and ASAP1-IT1, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01 compared with miR-509-3p + pcDNA. D Western blotting analysis on apoptosis-associated genes (Bax, Bcl-2, and cleaved-caspase-3) in A549 stem cells, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01 compared with miR-509-3p + pcDNA. E Colony formation assay on cell growth after 15 days culture, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01 compared with miR-509-3p + pcDNA. F qRT-PCR analysis of growth-associated genes (Cyclin A1, Cyclin B1, and PCNA) in A549 stem cells, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01 compared with miR-509-3p + pcDNA. G Tumor volume in nude mice at the indicated time after inoculation, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01 compared with miR-509-3p + pcDNA. H Photo of harvested tumors at the 24th day after inoculation. I Tumor weight at the 24th day after

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: EV5 Figure Induced dimerization of BAX and DRP1 induces apoptosis A Confocal microscopy of U2OS BAX/BAK DKO cells transfected with FKBP-mCherry-DRP1 (red) and FRB-EGFP-BAX (green) and stained with MitoTracker Deep Red FM (cyan). Images were acquired before (0 min) and after induction of BAX/DRP1 dimerization (5, 10 min). Scale bar 20 um. B-D Induced dimerization of DRP1 to itself (B), BAX to itself (C) or BAX to TOM20 (D) in U2OS BAX/BAK DKO cells transected with FKBP- and FRB-mCherry DRP1 (B, red), FKBP- and FRB-EGFP-BAX (C, green) or TOM20-mCherry-FKBP (red) and FRB-EGFP-BAX (green, D) before (0 min) and after induced dimerization (10 min). Mitochondria were stained using MitoTracker Deep Red FM or MitoSpy NIR (cyan) as indicated. Scale bar 20 um. Pearson's correlation coefficient was calculated between the induced dimerization signal of BAX/BAX, DRP1/DRP1 and BAX/TOM20, respectively, and mitochondria based on MitoTracker Deep Red TM signal as depicted below the images. E Induced dimerization of DRP1 and BAX mutants L63E, Delta19-37 or DeltaL1-2. Confocal microscopy of U2OS BAX/BAK DKO cells transfected with FKBP-mCherry-DRP1 (red) and mutant variants FRB-EGFP-BAX (green) as indicated and stained with MitoTracker Deep Red FM (cyan). Images were acquired 10 min after induction of BAX/DRP1 dimerization. Scale bar 20 um. All images are representative of n = 3 independent experiments. F-H Dimerization of BAX and DRP1 induces their translocation to mitochondria, exposure of the

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemical staining of cortical cell culture with antibodies against BCL-2 ( A , B ) and BAX ( D , E ), regulators of apoptosis, after 24-h incubation with 3 mug/mL of different-sized SeNPs without OGD/R ( A , D ) and 24 h after OGD/R ( B , E ). ( C , F )--Intensity levels of BCL-2 ( C ) and BAX ( F ) were determined by confocal imaging. We analyzed individual cells that had fluorescence of secondary antibodies. The quantative data reflecting the level of BCL-2 or BAX expression are presented as fluorescence intensity values in summary bar charts (mean +- SEM). The values were averaged by 150 cells for each column. The results obtained after immunostaining agree with the data of fluorescence presented in panels ( A , B , D , E ). Each value is the mean +- SE ( n >= 3, p < 0.05). Statistical significance was assessed using paired t -test. Black asterisks represent comparison of baseline expression levels after incubation with different sized nanoparticles versus control. Red asterisks represent comparison of expression level after incubation with different-sized SeNPs versus OGD/R-induced levels; n/s--data not significant ( p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 AS-IV treatment relieved I/R-induced apoptosis and regulated the Nrf2/HO-1 pathway in rat kidney tissues. (A) Apoptosis in the renal tissues of AKI rats as detected by TUNEL staining. Magnification x400. (B,C) Nrf2 and HO-1 were detected by IHC. Magnification x400. Bcl-2, Bax, Nrf2, and HO-1 expression were evaluated by qRT-PCR (D-G) and western blotting (H-L). # P