phosphor akt polyclonal antibody  (Proteintech)

Journal: iScience

Article Title: Maintaining Toll signaling in Drosophila brain is required to sustain autophagy for dopamine neuron survival

doi: 10.1016/j.isci.2024.108795

Figure Lengend Snippet: Overexpression of Toll, Spz, and Toll-Spz complex in S2 cells induces autophagy (A‒F) Detection of LC3-I, LC3-PE (LC3-II), and P62 proteins in S2 cells overexpressing Toll, Spz, and Toll-Spz complex by immunoblotting. S2 cells stably expressing RFP-GFP-LC3 were cultured in complete medium without treatment (control), or cultured in PBS instead of complete medium for 6 h (starved), or transfected with pMT-TIR-1-V5, pMT-TIR-7-V5 (A), pMT-Toll-1-V5, pMT-Toll-7-V5 (B), pMT-Spz-1-Flag, pMT-Spz-2-Flag, pMT-Spz-5-Flag, pMT-Spz-1FL-Flag (C), or pMT-GFP-V5 (D), or co-transfected with pMT-Toll-1-V5 or pMT-Toll-7-V5 with pMT-Spz-1-Flag, pMT-Spz-2-Flag, pMT-Spz-5-Flag, or pMT-Spz-1FL-Flag (E and F), then recombinant proteins were detected by mouse anti-V5 and mouse anti-Flag monoclonal antibodies, respectively. In these S2 cells, P62 protein was detected by anti-Ref(2)P antibody, LC3-I and LC3-II were detected by rabbit anti-LC3 polyclonal antibody, while tubulin was detected by mouse anti-tubulin monoclonal antibody. (G) Detection of Akt protein in S2 cells overexpressing Toll-Spz complex by immunoblotting. S2 cells stably expressing RFP-GFP-LC3 were treated as described above, total Akt (t-Akt) and phosphorylated Akt ( p -Akt) in cells were detected by rabbit anti-Akt and rabbit anti-phosphor-Akt ( p -Akt) polyclonal antibodies, respectively. Protein bands from at least 3 membranes were scanned for each protein using ImageJ. Data were represented as means ± SEM. Significant difference was determined by one way ANOVA followed by a Tukey’s multiple comparison tests using GraphPad Prism, with different letters indicating significant difference (p < 0.05) and identical letters for non-significant (p > 0.05). Significant difference was also determined by the student’s t test, ns for non-significant.

Article Snippet: Equal amounts of total proteins (50 μg) were loaded to 10%, 12% or 15% SDS-PAGE, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad), and the membrane was probed with one of the following primary antibodies: anti-V5-tag, anti-Flag-tag or anti-tubulin mouse monoclonal antibody, anti-LC3 rabbit polyclonal antibody (Sigma), anti-phosphor-Tor rabbit monoclonal antibody (Cell Signaling Technology, 5536), anti-Tor rabbit polyclonal antibody (ABclonal, A11354), anti-phosphor-S6K rabbit polyclonal antibody (ABclonal, AP0564), anti-S6K rabbit polyclonal antibody (ABclonal, A2190), anti-phosphor-Akt rabbit polyclonal antibody (Phospho- Drosophila Akt S505, Cell Signaling Technology, 4054), anti-Akt rabbit polyclonal antibody (Cell Signaling Technology, 9272), anti-DJ-1 rabbit monoclonal antibody (Cell Signaling Technology, 5933), anti-phosphor-JNK rabbit polyclonal antibody (Cell Signaling Technology, 9251), anti-PINK-1 rabbit monoclonal antibody (Cell Signaling Technology, 6946), anti-phosphor-GSK3 rabbit monoclonal antibody (Cell Signaling Technology, 9316), anti-Ref(2)P (P62) rabbit polyclonal antibody (abcam, ab178440), anti-Atg8 rabbit polyclonal antibody (abcam, ab109364), or anti-ubiquitin rabbit monoclonal antibody (PTM biolabs, PTM-1106).

Techniques: Over Expression, Western Blot, Stable Transfection, Expressing, Cell Culture, Control, Transfection, Recombinant, Bioprocessing, Comparison

Journal: iScience

Article Title: Maintaining Toll signaling in Drosophila brain is required to sustain autophagy for dopamine neuron survival

doi: 10.1016/j.isci.2024.108795

Figure Lengend Snippet: Toll-1 but not Toll-7 activated autophagy is dMyd88 dependent (A‒D) Detection of LC3-I, LC3-II, and P62 proteins in S2 cells treated with dsRNAs for dMyd88, Tube, Pelle, and Cactus by immunoblotting. S2 cells stably expressing RFP-GFP-LC3 were cultured in complete medium without treatment (control), or cultured in PBS instead of complete medium for 6 h (starved), or co-transfected with pMT-TIR-1-V5 or pMT-TIR-7-V5 with dsRNA for GFP (dsGFP), dMyd88 (dsMyd88) (A), Tube (dsTube) (B), Pelle (dsPelle) (C), or Cactus (dsCactus) (D), expression of TIR-1-V5 and TIR-7-V5 was detected by immunoblotting with mouse anti-V5 monoclonal antibody, LC3-I and LC3-II in these S2 cells were detected by rabbit anti-LC3 polyclonal antibody, P62 protein was detected by anti-Ref(2)P antibody, while tubulin was detected by mouse anti-tubulin monoclonal antibody. Data were represented as means ± SEM. For determination of relative protein level and significant differences, see Figure 1 legend.

Article Snippet: Equal amounts of total proteins (50 μg) were loaded to 10%, 12% or 15% SDS-PAGE, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad), and the membrane was probed with one of the following primary antibodies: anti-V5-tag, anti-Flag-tag or anti-tubulin mouse monoclonal antibody, anti-LC3 rabbit polyclonal antibody (Sigma), anti-phosphor-Tor rabbit monoclonal antibody (Cell Signaling Technology, 5536), anti-Tor rabbit polyclonal antibody (ABclonal, A11354), anti-phosphor-S6K rabbit polyclonal antibody (ABclonal, AP0564), anti-S6K rabbit polyclonal antibody (ABclonal, A2190), anti-phosphor-Akt rabbit polyclonal antibody (Phospho- Drosophila Akt S505, Cell Signaling Technology, 4054), anti-Akt rabbit polyclonal antibody (Cell Signaling Technology, 9272), anti-DJ-1 rabbit monoclonal antibody (Cell Signaling Technology, 5933), anti-phosphor-JNK rabbit polyclonal antibody (Cell Signaling Technology, 9251), anti-PINK-1 rabbit monoclonal antibody (Cell Signaling Technology, 6946), anti-phosphor-GSK3 rabbit monoclonal antibody (Cell Signaling Technology, 9316), anti-Ref(2)P (P62) rabbit polyclonal antibody (abcam, ab178440), anti-Atg8 rabbit polyclonal antibody (abcam, ab109364), or anti-ubiquitin rabbit monoclonal antibody (PTM biolabs, PTM-1106).

Techniques: Western Blot, Stable Transfection, Expressing, Cell Culture, Control, Transfection

Journal: iScience

Article Title: Maintaining Toll signaling in Drosophila brain is required to sustain autophagy for dopamine neuron survival

doi: 10.1016/j.isci.2024.108795

Figure Lengend Snippet: Both Toll-1 and Toll-7 activated autophagy requires PP2A activity (A and B) Expression of PP2A transcript in S2 cells after RNAi. (C) Detection of LC3-I, LC3-II and P62 proteins in S2 cells after RNAi of PP2A by immunoblotting. (D and E) Detection of PP2A activity in S2 cells treated with dsRNAs for several key genes in the Toll pathway. S2 cells stably expressing RFP-GFP-LC3 were cultured in complete medium without treatment (control), or cultured in PBS instead of complete medium for 6 h (starved), or co-transfected with pMT-TIR-1-V5 or pMT-TIR-7-V5 with dsRNA for GFP (dsGFP), PP2A (dsPP2A), dMyd88 (dsMyd88), Etc4 (dsEtc4), Traf6 (dsTraf6), Tube (dsTube), Pelle (dsPelle), or Cactus (dsCactus), RNAi efficiency of PP2A gene was determined by qRT-PCR (A, B), expression of TIR-1-V5 and TIR-7-V5 was detected by immunoblotting with mouse anti-V5 monoclonal antibody, LC3-I and LC3-II in these S2 cells were detected by rabbit anti-LC3 polyclonal antibody, P62 protein was detected by anti-Ref(2)P antibody, while tubulin was detected by mouse anti-tubulin monoclonal antibody (C). PP2A activity in these S2 cells was determined with the serine/threonine phosphatase assay system (D, E), and relative PP2A activity in the control samples was arbitrarily set as 1. Data were represented as means ± SEM. Significant difference was determined by the Student’s t test (A and B) and by one way ANOVA (C–E) (see Figure 1 legend).

Article Snippet: Equal amounts of total proteins (50 μg) were loaded to 10%, 12% or 15% SDS-PAGE, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad), and the membrane was probed with one of the following primary antibodies: anti-V5-tag, anti-Flag-tag or anti-tubulin mouse monoclonal antibody, anti-LC3 rabbit polyclonal antibody (Sigma), anti-phosphor-Tor rabbit monoclonal antibody (Cell Signaling Technology, 5536), anti-Tor rabbit polyclonal antibody (ABclonal, A11354), anti-phosphor-S6K rabbit polyclonal antibody (ABclonal, AP0564), anti-S6K rabbit polyclonal antibody (ABclonal, A2190), anti-phosphor-Akt rabbit polyclonal antibody (Phospho- Drosophila Akt S505, Cell Signaling Technology, 4054), anti-Akt rabbit polyclonal antibody (Cell Signaling Technology, 9272), anti-DJ-1 rabbit monoclonal antibody (Cell Signaling Technology, 5933), anti-phosphor-JNK rabbit polyclonal antibody (Cell Signaling Technology, 9251), anti-PINK-1 rabbit monoclonal antibody (Cell Signaling Technology, 6946), anti-phosphor-GSK3 rabbit monoclonal antibody (Cell Signaling Technology, 9316), anti-Ref(2)P (P62) rabbit polyclonal antibody (abcam, ab178440), anti-Atg8 rabbit polyclonal antibody (abcam, ab109364), or anti-ubiquitin rabbit monoclonal antibody (PTM biolabs, PTM-1106).

Techniques: Activity Assay, Expressing, Western Blot, Stable Transfection, Cell Culture, Control, Transfection, Quantitative RT-PCR, Phosphatase Assay

Journal: Current Research in Neurobiology

Article Title: Intranasal insulin attenuates hypoxia-ischemia-induced short-term sensorimotor behavioral disturbances, neuronal apoptosis, and brain damage in neonatal rats

doi: 10.1016/j.crneur.2023.100123

Figure Lengend Snippet: Insulin co-localizes with neuronal cells, leads to pAkt activation and subsequent reduction in HI-induced Cl-Caspase-3 positive cells in ipsilateral brain. Representative images following triple immunofluorescence (a-d) staining with Alex-546-Insulin (red), pAkt (green), and NeuN (blue) and double immunofluorescence staining (g-j) with NeuN (green) and Cl-Caspase-3 (red) of the ipsilateral hippocampal CA3 region at the dorsal hippocampal level at P11 following HI/Sham and immediate InInsulin/Veh treatment at P10 are shown, Scale bar = 100 μm. Quantification of pAkt and Cl-Caspase-3 positive cells in left (e, k) and right (f, l) hippocampal CA3 region at the dorsal hippocampal level, respectively are presented as box and whisker graphs. Two-way ANOVA followed by Post-hoc Holm-Sidak test, male and female pups were examined separately by the Kruskal-Wallis One Way Analysis of Variance on Ranks, followed by Post-hoc Student-Newman-Keuls test (for equal group sizes) or Dunn test (for equal group sizes), n = 4 pups/sex/group. Data are presented as median and range by the box and whisker plot. Dotted line represents the mean and symbol • represents outliers. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Polyclonal rabbit antibodies against phosphor-Akt (pAkt) and cleaved caspase 3 (Cl-Caspase 3) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Immunofluorescence, Staining, Double Immunofluorescence Staining, Whisker Assay

Journal: Cell

Article Title: Evidence That STK19 Is Not an NRAS-dependent Melanoma Driver

doi: 10.1016/j.cell.2020.04.014

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal to phosphor-AKT (ser473) , Cell signaling Technology , Cat#9271; RRID: AB_329825.

Techniques: Virus, Recombinant, Purification, Reverse Transcription, Software, Protease Inhibitor, Membrane, Transfection, Cloning, SYBR Green Assay, Mutagenesis, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Chlorzoxazone, a small molecule drug, augments immunosuppressive capacity of mesenchymal stem cells via modulation of FOXO3 phosphorylation

doi: 10.1038/s41419-020-2357-8

Figure Lengend Snippet: a Western blotting of p-FOXO3 and total FOXO3 influenced by CZ dose gradation. One gel was run and two blots were made in this image. One blot for total protein and the other for phosphorylated protein. The same loading control of GAPDH was used as Fig. . b Western blotting of p-FOXO3, total FOXO3, p-AKT, and p-ERK influenced by CZ, AKT, and/or ERK inhibitors. Two gels were run and two blots were made in this image. One blot for total protein and the other for phosphorylated protein. c Western blotting of p-FOXO3, total FOXO3, and p-AKT influenced by CZ and/or AKT activators. One gel was run and two blots were made in this image. One blot for total protein and the other for phosphorylated protein. All protein bands were visualized by chemiluminescent ECL reagent without antibody stripping procedure. d Immunofluorescence of total FOXO3 cellular location influenced by CZ and/or different kinase inhibitors and activators. Scale bars: 100 μm.

Article Snippet: After blocking in 5% bovine serum albumin (BSA) for 1 h at room temperature, the membranes were incubated with specific primary antibodies (FOXO3: ProteinTech, 10849-1-AP; phosphor-FOXO3 (p-FOXO3): Cell Signaling Technology (CST), 5538; ERK: CST, 4695; phosphor-ERK (p-ERK): CST, 4377; AKT: CST, 4691; phosphor-AKT (p-AKT): CST, 9277; GAPDH: ProteinTech, 10494-1-AP) at 4 °C overnight.

Techniques: Western Blot, Stripping Membranes, Immunofluorescence