foxo3a 75d8 rabbit mab  (Cell Signaling Technology Inc)

Journal: Advanced Science

Article Title: CAMKIIδ Reinforces Lipid Metabolism and Promotes the Development of B Cell Lymphoma

doi: 10.1002/advs.202409513

Figure Lengend Snippet: FOXO3A is a key substrate of CAMKIIδ. A) Proteomic analysis was conducted between Mutu CAMK2D wild‐type and knockout cells. The Venn diagram showed FOXO3A was one of the 18 proteins with both expression and phosphorylation changes. B) Representative western blots for FOXO3A protein levels in CAMK2D wild‐type (WT) and knockout (KO) cells. Data are presented as mean ± SD from three independent biological samples per group. ** p < 0.01. C) Representative western blot and D) IHC staining of FOXO3A between EµMYC; Camk2d wt/wt and EµMYC; Camk2d ‐/‐ mice. The bars represent 50µm. H score was determined by ImageJ. ** p < 0.01. E) Growth curve of Mutu CAMK2D knockout cells with either FOXO3A knockdown (SH) or scramble control (SCR). Data are presented as mean ± SD from four independent biological samples per group. F) (upper) Representative images of the electron microscope of lipid droplets in the indicated cells. The bars represent 2 µm. (lower) Confocal microscope imaging of BODIPY 493/503 staining in the indicated cells. Blue represents DAPI staining, and green represents BODIPY staining. The bars represent 10 µm. G) The median fluorescent intensity (MFI) of BODIPY 493/503 in the indicated cells was analyzed by flow cytometry. MFI was normalized by fluorescent mode. Representative overlays among the indicated cells were shown. Data are presented as mean ± SD from three technical replicates. H) Diagram of FOXO3A binding on IRF4 promoter region. I) The enrichment of FOXO3A on the IRF4 promoter region was examined by ChIP. The enriched DNA sequence was detected by qPCR. ** p < 0.01 in FOXO3A‐ChIP compared to IgG. All the results shown here, except panel A, are representative of three independent experiments. T‐test was used for analysis, unless otherwise indicated.

Article Snippet: The primary antibodies used were FOXO3A (75D8) Rabbit antibody (Cat#2497, CST, USA), CD45R (B220) Monoclonal Antibody (RA3‐6B2) (eBioscience, USA), Anti‐c‐Myc (Y69) antibody (Cat#ab32072, Abcam, USA), PLIN3 Rabbit anti‐Human Polyclonal Antibody (Cat#LS‐B2539, LSBio, USA).

Techniques: Knock-Out, Expressing, Phospho-proteomics, Western Blot, Immunohistochemistry, Knockdown, Control, Microscopy, Imaging, Staining, Flow Cytometry, Binding Assay, Sequencing

Journal: Advanced Science

Article Title: CAMKIIδ Reinforces Lipid Metabolism and Promotes the Development of B Cell Lymphoma

doi: 10.1002/advs.202409513

Figure Lengend Snippet: CAMKIIδ phosphorylates and destabilizes FOXO3A. A) Identification of FOXO3A S7 and S12 phosphorylation by CAMKIIδ from proteomic analysis. The mass shift of phosphorylation is 79.9663. Determination of S7: The theoretical molecular weight of SPA's three amino acid residues is 255.12. The calculation (b9−b6 = 1135.61−800.4249 = 355.185) matches the theoretical shift for SPA plus phosphorylation, calculated as (255.12+79.9663 = 355.086). Thus, it can be determined that the serine residue in SPA underwent phosphorylation modification. Determination of S12: The theoretical molecular weight of the peptide plus the charge is 2909.4. The actual mass shift across the entire peptide was calculated as (m/z × charge = 1023.118 × 3 = 3069.354), suggesting there are two phosphorylation sites on this peptide (2909.4+79.9663 × 2 = 3069.33). Furthermore, the y2−y1 fragment analysis indicates that S26 did not undergo phosphorylation modification. Since S7 was confirmed to be phosphorylated, it can be inferred that S12 is another phosphorylation site. B) HEK293 cells transfected with 3xFLAG‐FOXO3A, and CAMK2D‐HA or CAMK2G‐HA plasmids were co‐immunoprecipitated with HA‐beads; protein levels of 3xFLAG‐FOXO3A were analyzed by western blot. C) (left) In vitro kinase assay of CAMKIIδ on FOXO3A wild‐type and site mutants. HEK293 cells were transfected with 3xFLAG‐FOXO3A wild‐type, 3xFLAG‐FOXO3A S7A, S12A, and S7/12A plasmids. Proteins were then pulled down by FLAG beads, followed by an in vitro kinase assay supplied with Ca 2+ , calmodulin, and ATP. Total phosphorylated serine and threonine levels were detected by western blots. (right) In vitro kinase assay of CAMKIIδ and CAMKIIγ on FOXO3A protein. D) Radioisotope‐based in vitro kinase assay was performed on purified FOXO3A protein using 4 nM CAMKIIγ and 0.1 nM CAMKIIδ. E) Mutu CAMK2D wild‐type and knockout cells were treated with cycloheximide (CHX) for the indicated times. FOXO3A protein levels were then analyzed by western blot with Hsp90 as the loading control (left panel). Protein levels were measured with densitometric intensity. FOXO3A levels were quantified relative to Hsp90 levels and graphed as the percentage of remaining FOXO3A protein after treatment (right panel). F) Wild‐type BCL cells were treated with MG132, chloroquine, or DMSO control for indicated times, and FOXO3A expression was determined by western blot. GAPDH was used as a loading control. G) Venn diagram showing identified potential ubiquitylation regulator for FOXO3A by overlapping the MS results and online prediction. H) HEK293 cells transfected with vector control, WT or S7&12 mutant 3xFLAG‐FOXO3A were co‐immunoprecipitated with FLAG‐beads; protein levels of USP7 were analyzed by western blot. I) HEK293 cells overexpressing FLAG‐FOXO3A were transfected with HA‐ubiquitin and treated with 0.5µM USP7 inhibitor for 24 h. FOXO3A immunoprecipitated with FLAG beads, and HA‐ubiquitin was examined by western blot. All the results shown here, except panel A and G, are representative of three independent experiments. T‐test was used for analysis, unless otherwise indicated.

Article Snippet: The primary antibodies used were FOXO3A (75D8) Rabbit antibody (Cat#2497, CST, USA), CD45R (B220) Monoclonal Antibody (RA3‐6B2) (eBioscience, USA), Anti‐c‐Myc (Y69) antibody (Cat#ab32072, Abcam, USA), PLIN3 Rabbit anti‐Human Polyclonal Antibody (Cat#LS‐B2539, LSBio, USA).

Techniques: Phospho-proteomics, Molecular Weight, Residue, Modification, Transfection, Immunoprecipitation, Western Blot, In Vitro, Kinase Assay, Purification, Knock-Out, Control, Expressing, Plasmid Preparation, Mutagenesis, Ubiquitin Proteomics

Journal: Advanced Science

Article Title: CAMKIIδ Reinforces Lipid Metabolism and Promotes the Development of B Cell Lymphoma

doi: 10.1002/advs.202409513

Figure Lengend Snippet: TET inhibits CAMKIIδ activity and B cell lymphoma proliferation in vitro and in vivo. A) Molecular structure of Tetrandrine (TET). B) CETSA assay to determine the physical interaction between TET and CAMKIIδ. Cells were incubated with either DMSO or 10 µM TET for 1 h and cell lysates were incubated with gradient temperature from 45 °C to 52.5 °C. CAMKIIδ protein levels were detected by western blot assay. C) In vitro kinase assay of CAMKIIδ on FOXO3A in present with TET. HEK293 cells were transfected with 3xFLAG‐FOXO3A plasmid and FOXO3A protein was pulled down by FLAG‐beads. In vitro kinase assay was performed with purified CAMKIIδ protein on FLAG‐tagged FOXO3A, incubated with calcium, calmodulin, ATP, and indicated concentrations of TET. Total phosphorylated serine and threonine levels were detected by western blotting. D) Representative western blot of SU‐DHL‐6 CAMK2D WT and KO cells treated with indicated dosages of TET for 24 h. Tumor volume growth curve E) and tumor gross pictures F) of NSG mice treated with either PBS or TET. G) Tumor weight at the endpoint of the 32nd day. ** p < 0.01 versus PBS group. H) Representative western blot analysis of FOXO3A and CAMKIIδ protein levels in tumor tissues from PBS and TET treated groups. GAPDH is used as the loading control. I) Representative images of H&E and IHC staining of Ki67 and FOXO3A with sections of tumors from PBS and TET‐treated groups. The bar represents 50µm. J) IHC scores for CAMKIIδ and FOXO3A. K) Correlation of overall CAMKIIδ and nuclear FOXO3A levels. The P‐value was evaluated by Pearson Correlation assay. T‐test was used for analysis unless otherwise indicated. All the results shown here are representative of three independent experiments.

Article Snippet: The primary antibodies used were FOXO3A (75D8) Rabbit antibody (Cat#2497, CST, USA), CD45R (B220) Monoclonal Antibody (RA3‐6B2) (eBioscience, USA), Anti‐c‐Myc (Y69) antibody (Cat#ab32072, Abcam, USA), PLIN3 Rabbit anti‐Human Polyclonal Antibody (Cat#LS‐B2539, LSBio, USA).

Techniques: Activity Assay, In Vitro, In Vivo, Incubation, Western Blot, Kinase Assay, Transfection, Plasmid Preparation, Purification, Control, Immunohistochemistry, Two-Photon Excitation Fluorescence Cross-Correlation Assay

Journal: Pharmaceuticals

Article Title: IL-13Rα2 Is Involved in Resistance to Doxorubicin and Survival of Osteosarcoma Patients

doi: 10.3390/ph17111526

Figure Lengend Snippet: Figure 7. Effects of IL-13Rα2 on apoptosis of osteosarcoma cells under treatment with doxorubicin: (A) U2OS and KHOS/NP cells were transfected with vectors, followed by 0.2 µM doxorubicin treatment and Western blot for IL-13Rα2, FOXO3a, BAX, BCL2, cleaved caspase 3, and actin was performed. (B) Flow cytometric analysis for apoptosis of osteosarcoma cells under treatment with doxorubicin. U2OS and KHOS/NP cells transfected with vectors were treated with 0.2 µM doxoru- bicin for 24 h, stained with Annexin V and propidium iodide, and examined using flow cytometry. The right upper and right lower quadrants were considered apoptotic populations. * p < 0.05; *** p < 0.001; ns, not significant; EV, empty vector for wild-type IL-13Rα2; IL-13Rα2-OE, vector for wild-type IL-13Rα2; shControl, control vector for shRNA; shIL-13Rα2, vector for shRNA for IL-13Rα2; DOX, doxorubicin.

Article Snippet: The following antibodies were used in Western blotting: IL-13RA2/CD213a2 (E7U7B) (#85677, Cell Signaling Technology, Beverly, MA, USA), transforming growth factor β (TGF-β) (#3709, Cell Signaling Technology, Beverly, MA, USA), snail (#ab180714, Abcam, Cambridge, UK), cyclin D1 (#2922, Cell Signaling Technology, Beverly, MA, USA), BAX (#2774, Cell Signaling Technology, Beverly, MA, USA), BCL2 (D17C4) (#3498, Cell Signaling Technology, Beverly, MA, USA), forkhead box O3 (FOXO3a) (75D8) (#2497, Cell Signaling Technology, Beverly, MA, USA), cleaved caspase-3 (Asp175) (5A1E) (#9664, Cell Signaling Technology, Beverly, MA, USA), and actin (#sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Transfection, Western Blot, Staining, Flow Cytometry, Plasmid Preparation, Control, shRNA

Journal: Scientific Reports

Article Title: Blockage of Akt activation suppresses cadmium-induced renal tubular cellular damages through aggrephagy in HK-2 cells

doi: 10.1038/s41598-024-64579-3

Figure Lengend Snippet: Roles of Akt in cadmium exposure-induced RPTECs death. ( a ) Cells were incubated with 0.1% DMSO, 10 μM MK2206, or 10 μM Akti-1/2 for 1 h, then incubated with or without 25 μM CdCl 2 (Cd) for 35 h. The viability of cells was determined by trypan blue exclusion assay. Each value of trypan blue exclusion assay is the percentage of trypan blue-positive cells and reflects the mean ± SD of at least three experiments with duplicate assays. ( b , c ) Cells were incubated with or without 25 μM CdCl 2 for 6.5 h. Cells were stained with anti-Pericentrin (green), anti-Ubiquitin (Ub: red) antibodies, and Hoechst for DNA (blue). Percentages of the cells containing Ubiquitin puncta colocalizing with Pericentrin puncta were quantified ( c ). ( d , e ) Cells were incubated with 0.1% DMSO, 10 µM MK2206, or 10 µM Akti-1/2 for 6.5 h. Cells were stained with anti-Lamp1 (green), anti-LC3B (Red) antibodies, and Hoechst for DNA (blue) ( d ). The number of LC3B puncta in the cell were counted and listed in ( e ). The data are combined from at least three experiments and compared by the Mann–Whitney U test. The horizontal bars indicate the mean. ( f – i ) Cells were incubated with 0.2% DMSO, 10 μM MK2206, 10 µM Akti-1/2 or 40 μM Chloroquine for 4 h for western blotting. ( j–m ) Cells were incubated with 0.1% DMSO, 10 μM Akti-1/2 ( j , k ), 10 μM MK2206, or 0.5 μM Torin ( l , m ) for 1 h, then incubated with or without 25 μM CdCl 2 for 6.5 h for western blotting. ( n ) Cells were incubated with 0.1% DMSO or 200 ng/ml rapamycin for 6.5 h for western blotting. ( o ) Cells were incubated with or without 25 μM CdCl 2 for the indicated time for western blotting. The numbers (n) of cells examined are shown. Scale bars: 15 µm. Immunoblots shown are representative of at least three independent experiments. The displayed blots were cropped, and the full-length blots are shown in Supplementary Fig. . Results of densitometric analysis (mean ± SD) of at least three independent experiments. Arrows indicate the positions of P-FOXO3a ( j ) or P-TFEB S211 ( h , j ). * P < 0.05, ** P < 0.01, significant difference between the samples.

Article Snippet: Antibodies against LC3B, LC3B (D11) XP®, LC3B (E5Q2K), phospho-Akt (Thr308) (C31E5E), phospho-Akt (Ser473) (DQE) XP®, Akt (pan) (C67E7), phospho-p70 S6 kinase (Thr-389) (108D2), phospho-TFEB (Ser211) (EQS8N), phospho-TFEB (Ser122), TFEB, Cathepsin B (D1C7Y) XP®, Cathepsin D (E179), phospho-FoxO1 (Tr24)/FoxO3a (Thr32), FOXO3a (75D8), Vimentin (D21H3) XP®, and HDAC6 (D2E5) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Incubation, Trypan Blue Exclusion Assay, Staining, Ubiquitin Proteomics, MANN-WHITNEY, Western Blot

Journal: Scientific Reports

Article Title: Blockage of Akt activation suppresses cadmium-induced renal tubular cellular damages through aggrephagy in HK-2 cells

doi: 10.1038/s41598-024-64579-3

Figure Lengend Snippet: Effects of a calcineurin inhibitor (FK506) on TFEB/TFE3 phosphorylation status under cadmium stress. ( a ) HK-2 cells were incubated with 0.1% DMSO or 10–20 µM FK506 for 1 h, and then incubated with or without 25 µM CdCl 2 (Cd) for 2 h for western blotting. ( b , c ) HK-2 cells ( b ) and RPTECs ( c ) were incubated with 0.1% DMSO, 10 µM MK2206, or 0.5 µM Torin for 4 h and then incubated with or without 20 µM FK506 for 3 h for western blotting. ( d ) RPTECs were incubated with 0.1% DMSO or 100 nM okadaic acid for 1 h, and then incubated with or without 25 µM CdCl 2 for 2 h for western blotting. Immunoblots shown are representative of at least three independent experiments. The displayed blots were cropped, and the full-length blots are shown in Supplementary Fig. . Arrows indicate the positions of P-FOXO3a ( c ) or P-TFEB-S211 ( d ).

Article Snippet: Antibodies against LC3B, LC3B (D11) XP®, LC3B (E5Q2K), phospho-Akt (Thr308) (C31E5E), phospho-Akt (Ser473) (DQE) XP®, Akt (pan) (C67E7), phospho-p70 S6 kinase (Thr-389) (108D2), phospho-TFEB (Ser211) (EQS8N), phospho-TFEB (Ser122), TFEB, Cathepsin B (D1C7Y) XP®, Cathepsin D (E179), phospho-FoxO1 (Tr24)/FoxO3a (Thr32), FOXO3a (75D8), Vimentin (D21H3) XP®, and HDAC6 (D2E5) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Phospho-proteomics, Incubation, Western Blot

Journal: Immunity & Ageing : I & A

Article Title: Bifidobacterium bifidum and Lactobacillus paracasei alleviate sarcopenia and cognitive impairment in aged mice by regulating gut microbiota-mediated AKT, NF-κB, and FOXO3a signaling pathways

doi: 10.1186/s12979-023-00381-5

Figure Lengend Snippet: Effects of Bb, Lp, and LB on the myogenesis gene expression in the GA muscle of aged mice. a Effects on MuRF-1, MAFbx, p16, p-FOXO3a, FOXO3a, p-p65, p65, and β-actin expression, assessed by immunoblotting. b Effects on NF-κB + /CD11c + and MuRF1 + cell population, assessed by immunofluorescence staining. Effects on MuRF-1 ( c ), MAFbx/Atrogin-1 ( d ), TNF-α ( e ), and IL-6 expression ( f ), assessed by qPCR. Effects on PGC1α, MyHC, and β-action expression ( g , immunoblotted), GA muscle cell size ( h , H&E-stained), and MyHC-positive cell population ( i , stained with immunofluorescence-stained). Effects on MyHC ( j ), MyHC 2 A ( k ), MyHC 2X ( l ), MyHC 2B ( m ), MyoG ( n ), PGC1a ( o ), SIRT1 ( p ), and mtDNA ( q ) expression ( n ), assessed by qPCR. Bb, Lp, and LB (1 × 10 9 CFU/mouse/day) and Cr (75 mg/kg) were orally gavaged once a day (6 days in one week) for 8 weeks. Data are indicated as mean ± SD (n = 6). # p < 0.05 vs. Yg. * p < 0.05 vs. Vh/Ag

Article Snippet: The used antibodies are follows: phospho-Akt (Ser473) (193H12, Cell Signaling, Danvers, MA), Akt (11E7) (4685, Cell Signaling), muscle atrophy F-box gene (MAFbx, F-9, sc-166,806, Santa Cruz Biotechnology, Santa Cruz, CA), MuRF1(C-11) (sc-398,608, Santa Cruz Biotechnology), phospho-mTOR (ser2448) (2971, Cell Signaling), mammalian target of rapamycin (mTOR, 7C10, 2983,Cell Signaling), p-NF-κB-p65 (S536) (93H1, Cell Signaling), NF-κB-p65 (D14E12) (8242, Cell Signaling), myosin heavy chain (MyHc, B-5, sc-376,157, Santa Cruz Biotechnology), p16INK4A (E5F3Y, Cell signaling), p-FOXO3a (ser253, Cell Signaling), FOXO3a (75D8, Cell Signaling), β-actin (sc-47,778, Santa Cruz Biotechnology), and peroxisome proliferator-activated receptor gamma coactivator (PGC)1α (ab191838, Abcam).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Immunity & Ageing : I & A

Article Title: Bifidobacterium bifidum and Lactobacillus paracasei alleviate sarcopenia and cognitive impairment in aged mice by regulating gut microbiota-mediated AKT, NF-κB, and FOXO3a signaling pathways

doi: 10.1186/s12979-023-00381-5

Figure Lengend Snippet: Effects of Bb, Lp, and LB on the cognitive function in aged mice. a Effects on spontaneous alternation in the Y-maze task. Effects on hippocampal BDNF ( b ), TNF-α ( c ), IL-6 ( d ), IL-1β ( e ), and IL-10 ( f ) expression, TNF-α to IL-10 expression ratio ( g ), and IL-6 to IL-10 expression ratio ( h ), assessed by qPCR. Effects on BDNF + NeuN + ( i ) and NF-κB + Iba1 + cell populations ( j ). ( k ) Effects on p-FOXO3a and FOXO3a expression, assessed by immunoblotting. Effects on blood endotoxin ( l ) and corticosterone levels ( m ). Bb, Lp, and LB (1 × 10 9 CFU/mouse/day) and Cr (75 mg/kg) were orally gavaged once a day (6 days in one week) for 8 weeks. Data are indicated as mean ± SD ( n = 6). # p < 0.05 vs. Yg. * p < 0.05 vs. Vh/Ag

Article Snippet: The used antibodies are follows: phospho-Akt (Ser473) (193H12, Cell Signaling, Danvers, MA), Akt (11E7) (4685, Cell Signaling), muscle atrophy F-box gene (MAFbx, F-9, sc-166,806, Santa Cruz Biotechnology, Santa Cruz, CA), MuRF1(C-11) (sc-398,608, Santa Cruz Biotechnology), phospho-mTOR (ser2448) (2971, Cell Signaling), mammalian target of rapamycin (mTOR, 7C10, 2983,Cell Signaling), p-NF-κB-p65 (S536) (93H1, Cell Signaling), NF-κB-p65 (D14E12) (8242, Cell Signaling), myosin heavy chain (MyHc, B-5, sc-376,157, Santa Cruz Biotechnology), p16INK4A (E5F3Y, Cell signaling), p-FOXO3a (ser253, Cell Signaling), FOXO3a (75D8, Cell Signaling), β-actin (sc-47,778, Santa Cruz Biotechnology), and peroxisome proliferator-activated receptor gamma coactivator (PGC)1α (ab191838, Abcam).

Techniques: Expressing, Western Blot