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rrid bag3 primary antibody proteintech  (Proteintech)


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    Proteintech rrid bag3 primary antibody proteintech
    Rrid Bag3 Primary Antibody Proteintech, supplied by Proteintech, used in various techniques. Bioz Stars score: 97/100, based on 8066 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/primary+antibodies+%CE%B2+actin/beta+Actin+Monoclonal+antibody/pm41911062-140-7-11
    Average 97 stars, based on 8066 article reviews
    rrid bag3 primary antibody proteintech - by Bioz Stars, 2026-09
    97/100 stars

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    Expressing:

    Article Title: Expression, characterization and anti-colon cancer activity of recombinant ginseng peptides with amino acid tandem repeats.
    Article Snippet: Ginseng peptides, small molecule active ingredients in ginseng, are mainly extracted naturally or synthesized chemically, but high costs and difficulties hinder further research.. In this study, a ginseng hexapeptide FKEHGY, named antitumor peptide 0601 (AT0601) and its five tandem sequence repeats AT0605, were expressed in Bacillus subtilis WB600 for the first time, and the bioactivity study showed that the anticancer activity of AT0605 was even significantly higher than that of AT0601 for colon cancer CT26 cells, with IC50s of 16.82 ± 1.3 μM (48 h) and 855.57 ± 6.04 μM (48 h), respectively, i.e. AT0605 achieves the same inhibition rate for CT26 cells at a concentration 50 times lower than that of AT0601.. Similar to the ginseng peptide AT0601, recombinant AT0605 also inhibited cell growth by blocking cells in the G1 phase and activated the mitochondria-associated caspase pathway to induce apoptosis.

    Incubation:

    Article Title: Expression, characterization and anti-colon cancer activity of recombinant ginseng peptides with amino acid tandem repeats.
    Article Snippet: Ginseng peptides, small molecule active ingredients in ginseng, are mainly extracted naturally or synthesized chemically, but high costs and difficulties hinder further research.. In this study, a ginseng hexapeptide FKEHGY, named antitumor peptide 0601 (AT0601) and its five tandem sequence repeats AT0605, were expressed in Bacillus subtilis WB600 for the first time, and the bioactivity study showed that the anticancer activity of AT0605 was even significantly higher than that of AT0601 for colon cancer CT26 cells, with IC50s of 16.82 ± 1.3 μM (48 h) and 855.57 ± 6.04 μM (48 h), respectively, i.e. AT0605 achieves the same inhibition rate for CT26 cells at a concentration 50 times lower than that of AT0601.. Similar to the ginseng peptide AT0601, recombinant AT0605 also inhibited cell growth by blocking cells in the G1 phase and activated the mitochondria-associated caspase pathway to induce apoptosis.

    Article Title: Water-soluble polysaccharides derived from Poria cocos protect against LPS-induced renal injury through inhibiting the NF-κB-NOX4 signaling pathway.
    Article Snippet: .. Subsequently, the membranes were incubated secondary antibodies at room temperature for 1 h. Primary antibodies β-actin (Proteintech, 66009–1-Ig, 1:10000), NOX4 (Abclonal, #A23465, 1:1000), IκBα (Proteintech, 66418–1-Ig, 1:1000), p-IκBα (Proteintech, 82349–1-RR, 1:1000), and secondary antibodies (anti-mouse IgG-HRP and anti-rabbit IgG-HRP, 1:100000, Jackson ImmunoResearch) were used. .. The proteins were developed using the ECL Luminescence Kit (abs920, Absin) and analyzed with a Chem-Doc XRS system (Bio-Rad, Hercules, CA, USA).

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5 % skim milk for 2 h. Specific primary antibodies β-actin (15,204–1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Article Title: Structure-based discovery of a novel allosteric activator of ATG4B for the treatment of triple-negative breast cancer.
    Article Snippet: Triple-negative breast cancer (TNBC) is highly malignant and lacks effective therapeutic drugs.. ATG4B, a critical cysteine protease in autophagy, regulates LC3B processing and recycling to promote autophagosome formation, and its activation represents a promising therapeutic strategy for cancer.. Based on structure-based drug design, this study conducted virtual screening of the SPECS compound library and optimized the structure through structure-activity relationship analysis, obtaining a novel ATG4B allosteric activator, 16a.

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression.
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5% skim milk for 2 h. Specific primary antibodies β-actin (15,204−1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Lysis:

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5 % skim milk for 2 h. Specific primary antibodies β-actin (15,204–1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression.
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5% skim milk for 2 h. Specific primary antibodies β-actin (15,204−1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Protease Inhibitor:

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5 % skim milk for 2 h. Specific primary antibodies β-actin (15,204–1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression.
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5% skim milk for 2 h. Specific primary antibodies β-actin (15,204−1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Bicinchoninic Acid Protein Assay:

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5 % skim milk for 2 h. Specific primary antibodies β-actin (15,204–1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression.
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5% skim milk for 2 h. Specific primary antibodies β-actin (15,204−1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Isolation:

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5 % skim milk for 2 h. Specific primary antibodies β-actin (15,204–1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression.
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5% skim milk for 2 h. Specific primary antibodies β-actin (15,204−1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Membrane:

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5 % skim milk for 2 h. Specific primary antibodies β-actin (15,204–1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Article Title: Structure-based discovery of a novel allosteric activator of ATG4B for the treatment of triple-negative breast cancer.
    Article Snippet: Triple-negative breast cancer (TNBC) is highly malignant and lacks effective therapeutic drugs.. ATG4B, a critical cysteine protease in autophagy, regulates LC3B processing and recycling to promote autophagosome formation, and its activation represents a promising therapeutic strategy for cancer.. Based on structure-based drug design, this study conducted virtual screening of the SPECS compound library and optimized the structure through structure-activity relationship analysis, obtaining a novel ATG4B allosteric activator, 16a.

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression.
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5% skim milk for 2 h. Specific primary antibodies β-actin (15,204−1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Saline:

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5 % skim milk for 2 h. Specific primary antibodies β-actin (15,204–1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Article Title: Tanshinone IIA alleviates IL-1β-induced chondrocyte apoptosis and inflammation by regulating FBXO11 expression.
    Article Snippet: .. The collected cells and tissues were lysed in a RIPA lysis buffer mixed with protease inhibitor and phosphatase inhibitor (Roche, Switzerland) for 30 min. After detection with the BCA Protein Assay Kit (Beyotime), proteins were isolated on 10 % SDS-PAGE, loaded to a PVDF membrane (Millipore, USA), and sealed with 5% skim milk for 2 h. Specific primary antibodies β-actin (15,204−1-p, Proteintech), Cleaved caspase-3 (Asp175, Cell Signaling Technology, USA), FBXO11 (ab181801, Abcam), iNOS (ab178945, Abcam), PI3K (ab302958, Abcam), p-PI3K, AKT (ab8805, Abcam), p-AKT, p65 (ab111577, Abcam), and p-p65 were incubated overnight and washed in three-buffered saline containing 0.1 % Tween-20. .. The secondary antibody coupled with horseradish peroxidase was reacted for 6 h. Protein bands were developed by the Chemo Dox XRS system (Bio-Rad, USA), of which the optical density was calculated by ImageJ version 6.0.

    Blocking Assay:

    Article Title: Structure-based discovery of a novel allosteric activator of ATG4B for the treatment of triple-negative breast cancer.
    Article Snippet: Triple-negative breast cancer (TNBC) is highly malignant and lacks effective therapeutic drugs.. ATG4B, a critical cysteine protease in autophagy, regulates LC3B processing and recycling to promote autophagosome formation, and its activation represents a promising therapeutic strategy for cancer.. Based on structure-based drug design, this study conducted virtual screening of the SPECS compound library and optimized the structure through structure-activity relationship analysis, obtaining a novel ATG4B allosteric activator, 16a.



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    Cell Signaling Technology Inc β actin primary antibody
    A) RT-qPCR showed increased TLR3 mRNA expression for BEAS-2B transfected with a CA-FOXO1 plasmid compared to vector control (cells transfected with an empty plasmid); GAPDH was used as a housekeeping gene (n = 6). Representative Western blot (B) and densitometry analysis (C) of TLR3 expression for BEAS-2B transfected with CA-FOXO1 plasmid compared to vector control, <t>β-actin</t> was used as a loading control (n = 6). Statistical Analysis with t-test, **p < 0.01. D + E) Immunofluorescence staining for BEAS-2B transduced with CA-FOXO1 shows increased FOXO1 protein in the nucleus. FOXO1 (red) was detected using an anti-FOXO1 antibody with a red-fluorescent secondary antibody, F-actin (green) with phalloidin, and nuclei (blue) with DAPI. Images were taken with an Olympus IX81 epifluorescence microscope using a 20X objective lens. Volocity Analysis was used to quantify nuclear localization of FOXO1 by measuring the mean fluorescence intensity of FOXO1 staining colocalized with DAPI. For each group 40−60 cells per slide were analyzed. Statistical Analysis was conducted with ANOVA **** p < 0.001. BEAS-2B cells transduced with FOXO1 or scrambled shRNA lentivirus were analyzed by RT-qPCR for DDX58 (RIG-I, F), MAVS (G), and MYD88 (H) mRNA expression at baseline and after Poly(I:C) stimulation (8 h and 24 h). Expression was normalized to GAPDH and expressed relative to unstimulated scrambled controls (n = 3; ANOVA). (I) NHBE cells were infected with SARS-CoV-2 in the presence or absence of a FOXO1 inhibitor. Total RNA was collected 24 h post-infection, and viral RNA levels were quantified by qRT-PCR, normalized to ACTB, and expressed relative to mock-infected cells (n = 3; paired t-test).
    β Actin Primary Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rrid bag3 primary antibody proteintech  (Proteintech)
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    Proteintech rrid bag3 primary antibody proteintech
    A) RT-qPCR showed increased TLR3 mRNA expression for BEAS-2B transfected with a CA-FOXO1 plasmid compared to vector control (cells transfected with an empty plasmid); GAPDH was used as a housekeeping gene (n = 6). Representative Western blot (B) and densitometry analysis (C) of TLR3 expression for BEAS-2B transfected with CA-FOXO1 plasmid compared to vector control, <t>β-actin</t> was used as a loading control (n = 6). Statistical Analysis with t-test, **p < 0.01. D + E) Immunofluorescence staining for BEAS-2B transduced with CA-FOXO1 shows increased FOXO1 protein in the nucleus. FOXO1 (red) was detected using an anti-FOXO1 antibody with a red-fluorescent secondary antibody, F-actin (green) with phalloidin, and nuclei (blue) with DAPI. Images were taken with an Olympus IX81 epifluorescence microscope using a 20X objective lens. Volocity Analysis was used to quantify nuclear localization of FOXO1 by measuring the mean fluorescence intensity of FOXO1 staining colocalized with DAPI. For each group 40−60 cells per slide were analyzed. Statistical Analysis was conducted with ANOVA **** p < 0.001. BEAS-2B cells transduced with FOXO1 or scrambled shRNA lentivirus were analyzed by RT-qPCR for DDX58 (RIG-I, F), MAVS (G), and MYD88 (H) mRNA expression at baseline and after Poly(I:C) stimulation (8 h and 24 h). Expression was normalized to GAPDH and expressed relative to unstimulated scrambled controls (n = 3; ANOVA). (I) NHBE cells were infected with SARS-CoV-2 in the presence or absence of a FOXO1 inhibitor. Total RNA was collected 24 h post-infection, and viral RNA levels were quantified by qRT-PCR, normalized to ACTB, and expressed relative to mock-infected cells (n = 3; paired t-test).
    Rrid Bag3 Primary Antibody Proteintech, supplied by Proteintech, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti beta actin primary antibody  (Novus Biologicals)
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    Novus Biologicals anti beta actin primary antibody
    A) RT-qPCR showed increased TLR3 mRNA expression for BEAS-2B transfected with a CA-FOXO1 plasmid compared to vector control (cells transfected with an empty plasmid); GAPDH was used as a housekeeping gene (n = 6). Representative Western blot (B) and densitometry analysis (C) of TLR3 expression for BEAS-2B transfected with CA-FOXO1 plasmid compared to vector control, <t>β-actin</t> was used as a loading control (n = 6). Statistical Analysis with t-test, **p < 0.01. D + E) Immunofluorescence staining for BEAS-2B transduced with CA-FOXO1 shows increased FOXO1 protein in the nucleus. FOXO1 (red) was detected using an anti-FOXO1 antibody with a red-fluorescent secondary antibody, F-actin (green) with phalloidin, and nuclei (blue) with DAPI. Images were taken with an Olympus IX81 epifluorescence microscope using a 20X objective lens. Volocity Analysis was used to quantify nuclear localization of FOXO1 by measuring the mean fluorescence intensity of FOXO1 staining colocalized with DAPI. For each group 40−60 cells per slide were analyzed. Statistical Analysis was conducted with ANOVA **** p < 0.001. BEAS-2B cells transduced with FOXO1 or scrambled shRNA lentivirus were analyzed by RT-qPCR for DDX58 (RIG-I, F), MAVS (G), and MYD88 (H) mRNA expression at baseline and after Poly(I:C) stimulation (8 h and 24 h). Expression was normalized to GAPDH and expressed relative to unstimulated scrambled controls (n = 3; ANOVA). (I) NHBE cells were infected with SARS-CoV-2 in the presence or absence of a FOXO1 inhibitor. Total RNA was collected 24 h post-infection, and viral RNA levels were quantified by qRT-PCR, normalized to ACTB, and expressed relative to mock-infected cells (n = 3; paired t-test).
    Anti Beta Actin Primary Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Journal: Frontiers in Physiology

    Article Title: Transcriptomic analysis and experimental verification reveal the involvement of PI3K/AKT signaling pathway in high-altitude cognitive dysfunction

    doi: 10.3389/fphys.2026.1781613

    Figure Lengend Snippet: Primer sequences for the genes observed in qRT-PCR.

    Article Snippet: Primary antibody against β -actin were purchased from Zhongshan Golden Bridge Biological Technology Co., Ltd. (Beijing, China).

    Techniques:

    Effects of HH on the BBB integrity in mouse hippocampus. (A) Representative images of EB extravasation in the hippocampus. (B) Quantitative analysis of EB leakage. (C) Representative western blots of ZO-1 and occludin. Quantitative analysis of the ZO-1/β-actin (D) and occludin/β-actin (E) ratios. Data are presented as mean ± SD. Statistical comparisons were performed by Student’s t test. For EB, n = 6 per group; for Western blot analysis, n = 3 per group, ** p < 0.01 vs. Con group.

    Journal: Frontiers in Physiology

    Article Title: Transcriptomic analysis and experimental verification reveal the involvement of PI3K/AKT signaling pathway in high-altitude cognitive dysfunction

    doi: 10.3389/fphys.2026.1781613

    Figure Lengend Snippet: Effects of HH on the BBB integrity in mouse hippocampus. (A) Representative images of EB extravasation in the hippocampus. (B) Quantitative analysis of EB leakage. (C) Representative western blots of ZO-1 and occludin. Quantitative analysis of the ZO-1/β-actin (D) and occludin/β-actin (E) ratios. Data are presented as mean ± SD. Statistical comparisons were performed by Student’s t test. For EB, n = 6 per group; for Western blot analysis, n = 3 per group, ** p < 0.01 vs. Con group.

    Article Snippet: Primary antibody against β -actin were purchased from Zhongshan Golden Bridge Biological Technology Co., Ltd. (Beijing, China).

    Techniques: Western Blot

    Effects of HH on the expression of genes and proteins related to PI3K/AKT signaling pathway in mouse hippocampus. (A) The relative mRNA expression levels of Kdr , Spp1 , Vwf , and Vegfa screened via qRT-PCR. (B) Representative western blots of p-PI3K, PI3K, p-AKT, and AKT. Quantitative analysis of the p-PI3K/PI3K (C) and p-AKT/AKT (D) ratios. (E) Representative western blots of Nrf2, HO-1, p-NF-κB, and NF-κB. Quantitative analysis of the Nrf2/β-actin (F) , HO-1/β-actin (G) , and p-NF-κB/NF-κB (H) ratios. Data are presented as mean ± SD. Statistical comparisons were performed by Student’s t test. For qRT-PCR, n = 6 per group; for Western blot analysis, n = 3 per group, * p < 0.05, ** p < 0.01 vs. Con group.

    Journal: Frontiers in Physiology

    Article Title: Transcriptomic analysis and experimental verification reveal the involvement of PI3K/AKT signaling pathway in high-altitude cognitive dysfunction

    doi: 10.3389/fphys.2026.1781613

    Figure Lengend Snippet: Effects of HH on the expression of genes and proteins related to PI3K/AKT signaling pathway in mouse hippocampus. (A) The relative mRNA expression levels of Kdr , Spp1 , Vwf , and Vegfa screened via qRT-PCR. (B) Representative western blots of p-PI3K, PI3K, p-AKT, and AKT. Quantitative analysis of the p-PI3K/PI3K (C) and p-AKT/AKT (D) ratios. (E) Representative western blots of Nrf2, HO-1, p-NF-κB, and NF-κB. Quantitative analysis of the Nrf2/β-actin (F) , HO-1/β-actin (G) , and p-NF-κB/NF-κB (H) ratios. Data are presented as mean ± SD. Statistical comparisons were performed by Student’s t test. For qRT-PCR, n = 6 per group; for Western blot analysis, n = 3 per group, * p < 0.05, ** p < 0.01 vs. Con group.

    Article Snippet: Primary antibody against β -actin were purchased from Zhongshan Golden Bridge Biological Technology Co., Ltd. (Beijing, China).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    SG formation is dependent on the PERK/eIF2α signaling pathway. CFBE cells were pretreated with DMSO, ISRIB or GSK and then exposed to 15% HBSS or smoke extract for 2 h. IF imaging or immunoblotting were then performed. ( A , B ) IF imaging showed that endogenous G3BP1 ( A ) and p-eIF2α ( B ) are homogenously distributed in the cell cytoplasm under control conditions (DMSO + HBSS). ( C ) Smoke exposure induced robust SG formation (white arrows) and ( D ) a significant increase in cytoplasmic eIF2α phosphorylation (p-eIF2α). ( E , F ) Pretreating cells with ISRIB, an inhibitor of p-eIF2α function, at 200 nM for 1 h prior to smoke extract exposure ( E ) completely abolished SG formation, establishing causality ( F ) without reducing p-eIF2α abundance. ( G , H ) Inhibiting p-PERK by pretreating cells with 2 µM GSK for 1 h prior to smoke extract exposure ( G ) completely blocked SG formation and ( H ) abrogated the increase in p-eIF2α levels, indicating that SG formation is PERK/eIF2α-dependent. ( I ) Quantitative nucleus-based image analysis of mean cytoplasmic p-eIF2α levels demonstrated a significant increase in cells’ response to smoke extract exposure as demonstrated by the 3-fold increase in cytoplasmic p-eIF2α fluorescence intensity (IF DMSO+HBSS = 724 ± 25, N DMSO+HBSS = 4, n DMSO+HBSS = 24; IF DMSO+Smoke = 1919 ± 48, N DMSO+Smoke = 16, n DMSO+Smoke = 120 (4 outliers were identified and excluded)). The analysis also showed that ISRIB pretreatment induced further accumulation and a significant increase in cytoplasmic p-eIF2α levels in response to smoke exposure (IF ISRIB+Smoke = 2464 ± 57, N ISRIB+Smoke = 7, n ISRIB+Smoke = 54). GSK pretreatment, on the other hand, significantly attenuated p-eIF2α increase in response to smoke exposure (IF GSK+Smoke = 1187 ± 34, N GSK+Smoke = 11, n GSK+Smoke = 82) without fully returning to the DMSO + HBSS baseline. ( J ) Quantitative nucleus-based image analysis of mean cytoplasmic p-eIF2α levels showed that pretreating cells with DMSO (drug vehicle control) did not induce significant change in cytoplasmic p-eIF2α levels under HBSS or smoke exposure conditions (IF HBSS = 725 ± 13, N HBSS = 18, n HBSS = 132 (1 outlier was identified and excluded); IF Smoke = 2249 ± 62, N Smoke = 19, n Smoke = 142). The nonparametric Kruskal–Wallis test was used to calculate significance in ( I , J ). ns: not significant, **: p = 0.01 and ****: p < 0.0001. Each ROI is an independent biological sample. ( K – N ) immunoblotting demonstrated a significant 5- to 7-fold increase in cellular p-eIF2α level in response to smoke exposure, with ISRIB pretreatment increasing this level non-significantly to 10-fold (N = 4, IB DMSO+HBSS = 1, IB DMSO+Smoke = 7 ± 1, IB ISRIB+Smoke = 10 ± 2). GSK pretreatment reduced p-eIF2α levels significantly but not to the DMSO + HBSS basal level (N = 5, IB DMSO+HBSS = 1, IB DMSO+Smoke = 5.1 ± 0.5, IB GSK+Smoke = 2.8 ± 0.5). Immunoblotting data were normalized to β-Actin protein level and then to the corresponding vehicle control. See also . Brown–Forsythe and Welch ANOVA tests were used to calculate significance in ( M , N ), with nd = no discovery and *: p < 0.03. Data are presented as mean ± SEM. Confocal image scale bar = 12.8 µm. Abbreviations: SEM = standard error of mean, SG = stress granule, ROI = region of interest, IF = immunofluorescence, IB = immunoblotting, N = the total number of independent immunofluorescence experiments and n = total number of technical replicates (or analyzed ROIs) in all immunofluorescence experiments (n = N × number of technical replicates per experiment). Western blot original images can be found in .

    Journal: Biomolecules

    Article Title: Cigarette Smoke Induces Canonical Stress Granule Formation in Human Bronchial Epithelial Cells in Reactive Oxygen Species- and PERK-Dependent Manners

    doi: 10.3390/biom16040615

    Figure Lengend Snippet: SG formation is dependent on the PERK/eIF2α signaling pathway. CFBE cells were pretreated with DMSO, ISRIB or GSK and then exposed to 15% HBSS or smoke extract for 2 h. IF imaging or immunoblotting were then performed. ( A , B ) IF imaging showed that endogenous G3BP1 ( A ) and p-eIF2α ( B ) are homogenously distributed in the cell cytoplasm under control conditions (DMSO + HBSS). ( C ) Smoke exposure induced robust SG formation (white arrows) and ( D ) a significant increase in cytoplasmic eIF2α phosphorylation (p-eIF2α). ( E , F ) Pretreating cells with ISRIB, an inhibitor of p-eIF2α function, at 200 nM for 1 h prior to smoke extract exposure ( E ) completely abolished SG formation, establishing causality ( F ) without reducing p-eIF2α abundance. ( G , H ) Inhibiting p-PERK by pretreating cells with 2 µM GSK for 1 h prior to smoke extract exposure ( G ) completely blocked SG formation and ( H ) abrogated the increase in p-eIF2α levels, indicating that SG formation is PERK/eIF2α-dependent. ( I ) Quantitative nucleus-based image analysis of mean cytoplasmic p-eIF2α levels demonstrated a significant increase in cells’ response to smoke extract exposure as demonstrated by the 3-fold increase in cytoplasmic p-eIF2α fluorescence intensity (IF DMSO+HBSS = 724 ± 25, N DMSO+HBSS = 4, n DMSO+HBSS = 24; IF DMSO+Smoke = 1919 ± 48, N DMSO+Smoke = 16, n DMSO+Smoke = 120 (4 outliers were identified and excluded)). The analysis also showed that ISRIB pretreatment induced further accumulation and a significant increase in cytoplasmic p-eIF2α levels in response to smoke exposure (IF ISRIB+Smoke = 2464 ± 57, N ISRIB+Smoke = 7, n ISRIB+Smoke = 54). GSK pretreatment, on the other hand, significantly attenuated p-eIF2α increase in response to smoke exposure (IF GSK+Smoke = 1187 ± 34, N GSK+Smoke = 11, n GSK+Smoke = 82) without fully returning to the DMSO + HBSS baseline. ( J ) Quantitative nucleus-based image analysis of mean cytoplasmic p-eIF2α levels showed that pretreating cells with DMSO (drug vehicle control) did not induce significant change in cytoplasmic p-eIF2α levels under HBSS or smoke exposure conditions (IF HBSS = 725 ± 13, N HBSS = 18, n HBSS = 132 (1 outlier was identified and excluded); IF Smoke = 2249 ± 62, N Smoke = 19, n Smoke = 142). The nonparametric Kruskal–Wallis test was used to calculate significance in ( I , J ). ns: not significant, **: p = 0.01 and ****: p < 0.0001. Each ROI is an independent biological sample. ( K – N ) immunoblotting demonstrated a significant 5- to 7-fold increase in cellular p-eIF2α level in response to smoke exposure, with ISRIB pretreatment increasing this level non-significantly to 10-fold (N = 4, IB DMSO+HBSS = 1, IB DMSO+Smoke = 7 ± 1, IB ISRIB+Smoke = 10 ± 2). GSK pretreatment reduced p-eIF2α levels significantly but not to the DMSO + HBSS basal level (N = 5, IB DMSO+HBSS = 1, IB DMSO+Smoke = 5.1 ± 0.5, IB GSK+Smoke = 2.8 ± 0.5). Immunoblotting data were normalized to β-Actin protein level and then to the corresponding vehicle control. See also . Brown–Forsythe and Welch ANOVA tests were used to calculate significance in ( M , N ), with nd = no discovery and *: p < 0.03. Data are presented as mean ± SEM. Confocal image scale bar = 12.8 µm. Abbreviations: SEM = standard error of mean, SG = stress granule, ROI = region of interest, IF = immunofluorescence, IB = immunoblotting, N = the total number of independent immunofluorescence experiments and n = total number of technical replicates (or analyzed ROIs) in all immunofluorescence experiments (n = N × number of technical replicates per experiment). Western blot original images can be found in .

    Article Snippet: The hFABTM Rhodamine β-Actin and GAPDH primary antibodies were used for immunoblotting experiments (Bio-Rad).

    Techniques: Imaging, Western Blot, Control, Phospho-proteomics, Fluorescence, Immunofluorescence

    Reactive oxygen species activate the PERK/eIF2α signaling pathway and induce SG formation in response to smoke exposure. CFBE cells were pretreated with H 2 O or 5 mM NAC for 15 min and then exposed to 15% HBSS or smoke extract for 2 h. IF imaging and immunoblotting analyses were then performed. ( A – C ) Immunoblotting (IB) demonstrated a significant 7-fold increase in cellular p-eIF2α level in response to smoke exposure which was fully abolished upon pretreating cells with NAC (IB H2O+HBSS = 1, IB H2O+Smoke = 7.1 ± 0.9, IB NAC+Smoke = 1.4 ± 0.3; N H2O+HBSS = N H2O+Smoke = 36, N NAC+Smoke = 10). Additionally, immunoblotting showed that smoke exposure increased p-PERK level by 2.8-fold, which is also completely abolished by NAC pretreatment (IB H2O+HBSS = 1, IB H2O+Smoke = 2.8 ± 0.3, IB NAC+Smoke = 1.3 ± 0.2; N H2O+HBSS = 41, N H2O+Smoke = 40, N NAC+Smoke = 14). IB data were normalized to β-Actin protein level and then to the corresponding vehicle control. See also . ( D , E ) Confocal IF imaging showed that ( D ) endogenous G3BP1 and ( E ) p-eIF2α were homogenously distributed in the cell cytoplasm under control conditions (H 2 O + HBSS). ( F , G ) IF imaging showed that smoke extract exposure induced SG formation (white arrows) and a marked increase in cytoplasmic p-eIF2α abundance. ( H , I ) Pretreating cells with the ROS scavenger NAC prior to smoke extract exposure completely abolished SG formation and inhibited the increase in p-eIF2α levels, establishing that SG formation is ROS-driven in p-eIF2α-dependent manner. ( J ) Quantitative nucleus-based image analysis showed that NAC pretreatment significantly attenuated the significant 3-fold increase in cytoplasmic p-eIF2α level in response to smoke extract exposure (IF H2O+HBSS = 700 ± 9, IF H2O+Smoke = 2485 ± 75, IF NAC+Smoke = 899 ± 28; N H2O+HBSS = 5, n H2O+HBSS = 30; N H2O+Smoke = 8, n H2O+Smoke = 47 (1 outlier was identified and excluded); N NAC+Smoke = 9, n NAC+Smoke = 54). Each ROI is an independent biological sample. The nonparametric Kruskal–Wallis test was used to calculate significance in ( B , C , J ). ns: not significant, *: p < 0.03, **: p = 0.0095, ***: p = 0.0002, and ****: p < 0.0001. Data are presented as mean ± SEM. Confocal image scale bar = 12.8 µm. Abbreviations: SEM = standard error of mean, SG = stress granule, ROI for region of interest, IF = immunofluorescence, IB = immunoblotting, N = the total number of independent immunofluorescence experiments, and n = the total number of technical replicates (or analyzed ROIs) in all immunofluorescence experiments (n = N × number of technical replicates per experiment). Western blot original images can be found in .

    Journal: Biomolecules

    Article Title: Cigarette Smoke Induces Canonical Stress Granule Formation in Human Bronchial Epithelial Cells in Reactive Oxygen Species- and PERK-Dependent Manners

    doi: 10.3390/biom16040615

    Figure Lengend Snippet: Reactive oxygen species activate the PERK/eIF2α signaling pathway and induce SG formation in response to smoke exposure. CFBE cells were pretreated with H 2 O or 5 mM NAC for 15 min and then exposed to 15% HBSS or smoke extract for 2 h. IF imaging and immunoblotting analyses were then performed. ( A – C ) Immunoblotting (IB) demonstrated a significant 7-fold increase in cellular p-eIF2α level in response to smoke exposure which was fully abolished upon pretreating cells with NAC (IB H2O+HBSS = 1, IB H2O+Smoke = 7.1 ± 0.9, IB NAC+Smoke = 1.4 ± 0.3; N H2O+HBSS = N H2O+Smoke = 36, N NAC+Smoke = 10). Additionally, immunoblotting showed that smoke exposure increased p-PERK level by 2.8-fold, which is also completely abolished by NAC pretreatment (IB H2O+HBSS = 1, IB H2O+Smoke = 2.8 ± 0.3, IB NAC+Smoke = 1.3 ± 0.2; N H2O+HBSS = 41, N H2O+Smoke = 40, N NAC+Smoke = 14). IB data were normalized to β-Actin protein level and then to the corresponding vehicle control. See also . ( D , E ) Confocal IF imaging showed that ( D ) endogenous G3BP1 and ( E ) p-eIF2α were homogenously distributed in the cell cytoplasm under control conditions (H 2 O + HBSS). ( F , G ) IF imaging showed that smoke extract exposure induced SG formation (white arrows) and a marked increase in cytoplasmic p-eIF2α abundance. ( H , I ) Pretreating cells with the ROS scavenger NAC prior to smoke extract exposure completely abolished SG formation and inhibited the increase in p-eIF2α levels, establishing that SG formation is ROS-driven in p-eIF2α-dependent manner. ( J ) Quantitative nucleus-based image analysis showed that NAC pretreatment significantly attenuated the significant 3-fold increase in cytoplasmic p-eIF2α level in response to smoke extract exposure (IF H2O+HBSS = 700 ± 9, IF H2O+Smoke = 2485 ± 75, IF NAC+Smoke = 899 ± 28; N H2O+HBSS = 5, n H2O+HBSS = 30; N H2O+Smoke = 8, n H2O+Smoke = 47 (1 outlier was identified and excluded); N NAC+Smoke = 9, n NAC+Smoke = 54). Each ROI is an independent biological sample. The nonparametric Kruskal–Wallis test was used to calculate significance in ( B , C , J ). ns: not significant, *: p < 0.03, **: p = 0.0095, ***: p = 0.0002, and ****: p < 0.0001. Data are presented as mean ± SEM. Confocal image scale bar = 12.8 µm. Abbreviations: SEM = standard error of mean, SG = stress granule, ROI for region of interest, IF = immunofluorescence, IB = immunoblotting, N = the total number of independent immunofluorescence experiments, and n = the total number of technical replicates (or analyzed ROIs) in all immunofluorescence experiments (n = N × number of technical replicates per experiment). Western blot original images can be found in .

    Article Snippet: The hFABTM Rhodamine β-Actin and GAPDH primary antibodies were used for immunoblotting experiments (Bio-Rad).

    Techniques: Imaging, Western Blot, Control, Immunofluorescence

    A) RT-qPCR showed increased TLR3 mRNA expression for BEAS-2B transfected with a CA-FOXO1 plasmid compared to vector control (cells transfected with an empty plasmid); GAPDH was used as a housekeeping gene (n = 6). Representative Western blot (B) and densitometry analysis (C) of TLR3 expression for BEAS-2B transfected with CA-FOXO1 plasmid compared to vector control, β-actin was used as a loading control (n = 6). Statistical Analysis with t-test, **p < 0.01. D + E) Immunofluorescence staining for BEAS-2B transduced with CA-FOXO1 shows increased FOXO1 protein in the nucleus. FOXO1 (red) was detected using an anti-FOXO1 antibody with a red-fluorescent secondary antibody, F-actin (green) with phalloidin, and nuclei (blue) with DAPI. Images were taken with an Olympus IX81 epifluorescence microscope using a 20X objective lens. Volocity Analysis was used to quantify nuclear localization of FOXO1 by measuring the mean fluorescence intensity of FOXO1 staining colocalized with DAPI. For each group 40−60 cells per slide were analyzed. Statistical Analysis was conducted with ANOVA **** p < 0.001. BEAS-2B cells transduced with FOXO1 or scrambled shRNA lentivirus were analyzed by RT-qPCR for DDX58 (RIG-I, F), MAVS (G), and MYD88 (H) mRNA expression at baseline and after Poly(I:C) stimulation (8 h and 24 h). Expression was normalized to GAPDH and expressed relative to unstimulated scrambled controls (n = 3; ANOVA). (I) NHBE cells were infected with SARS-CoV-2 in the presence or absence of a FOXO1 inhibitor. Total RNA was collected 24 h post-infection, and viral RNA levels were quantified by qRT-PCR, normalized to ACTB, and expressed relative to mock-infected cells (n = 3; paired t-test).

    Journal: PLOS One

    Article Title: FOXO1 transcription factor modulates airway epithelial responses to viral infection

    doi: 10.1371/journal.pone.0345169

    Figure Lengend Snippet: A) RT-qPCR showed increased TLR3 mRNA expression for BEAS-2B transfected with a CA-FOXO1 plasmid compared to vector control (cells transfected with an empty plasmid); GAPDH was used as a housekeeping gene (n = 6). Representative Western blot (B) and densitometry analysis (C) of TLR3 expression for BEAS-2B transfected with CA-FOXO1 plasmid compared to vector control, β-actin was used as a loading control (n = 6). Statistical Analysis with t-test, **p < 0.01. D + E) Immunofluorescence staining for BEAS-2B transduced with CA-FOXO1 shows increased FOXO1 protein in the nucleus. FOXO1 (red) was detected using an anti-FOXO1 antibody with a red-fluorescent secondary antibody, F-actin (green) with phalloidin, and nuclei (blue) with DAPI. Images were taken with an Olympus IX81 epifluorescence microscope using a 20X objective lens. Volocity Analysis was used to quantify nuclear localization of FOXO1 by measuring the mean fluorescence intensity of FOXO1 staining colocalized with DAPI. For each group 40−60 cells per slide were analyzed. Statistical Analysis was conducted with ANOVA **** p < 0.001. BEAS-2B cells transduced with FOXO1 or scrambled shRNA lentivirus were analyzed by RT-qPCR for DDX58 (RIG-I, F), MAVS (G), and MYD88 (H) mRNA expression at baseline and after Poly(I:C) stimulation (8 h and 24 h). Expression was normalized to GAPDH and expressed relative to unstimulated scrambled controls (n = 3; ANOVA). (I) NHBE cells were infected with SARS-CoV-2 in the presence or absence of a FOXO1 inhibitor. Total RNA was collected 24 h post-infection, and viral RNA levels were quantified by qRT-PCR, normalized to ACTB, and expressed relative to mock-infected cells (n = 3; paired t-test).

    Article Snippet: Primary mouse anti-β-actin mAb (Santa Cruz Biotechnology, SC-69679) and IRdye-conjugated donkey anti-mouse IgG (LI-COR, Lincoln, Neb) were used as a loading control.

    Techniques: Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Immunofluorescence, Staining, Transduction, Microscopy, Fluorescence, shRNA, Infection