rabbit polyclonal antibody against phospho pi3k  (Cell Signaling Technology Inc)

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Citrus unshiu Peel Attenuates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice due to Modulation of the PI3K/Akt Signaling Pathway and MAPK and NF- κ B

doi: 10.1155/2022/4041402

Figure Lengend Snippet: Citrus unshiu peel modulated the PI3K/Akt signaling pathway. Control: control mice, DSS: distilled water administered to ulcerative colitis mice, SASP: sulfasalazine 100 mg/kg body weight administered to ulcerative colitis mice, CUPL: CUP 100 mg/kg body weight administered to ulcerative colitis mice, and CUPH: CUP 200 mg/kg body weight administered to ulcerative colitis mice. Data are mean ± SD ( n = 8). Significance: ### p < 0.001 vs. control mice, ∗∗∗ p < 0.001 vs. DSS mice.

Article Snippet: Rabbit polyclonal antibodies against p-PI3K (#4228), Akt (1 : 1,000, #9272), p-Akt (#9275), and p38 MAPK (#9212) and mouse polyclonal antibodies against c-Jun (1 : 1,000, #2315) were purchased from Cell Signaling Technology, Inc. (Danvers, MN, USA).

Techniques: Control

Journal: REPRODUCTION

Article Title: Protein kinase A and protein kinase Cα/PPP1CC2 play opposing roles in the regulation of phosphatidylinositol 3-kinase activation in bovine sperm

doi: 10.1530/rep-09-0314

Figure Lengend Snippet: Figure 1 PRKCA is inhibited and PI3K is activated during sperm capacitation. Bovine sperm was incubated for 4 or 7 h in the mTALP capacitation medium (A–E) or NKM medium (non-capacitation medium) (A and B). (A) Capacitated or non-capacitated sperm proteins were extracted at the indicated times and western blotted as described in ‘Materials and Methods’. p-(Tyr467)-PI3K, p-(Ser657)-PRKCA, and PRKCA were detected using specific antibodies. (B) Capacitated or non-capacitated sperm proteins were extracted at the indicated times and western blotted. p-(Thr497)-PRKCA and PRKCA were detected using specific antibody. (C) PI3K was detected in sperm extracts at the indicated times using specific antibody. (A–C) b-Actin antibody was used to equalize the loading quantities. (D) p-(Tyr467)-PI3K, PRKCA, p-(Thr497)-PRKCA, or p-(Ser657)-PRKCA were detected at the indicated times, using specific antibodies on western blotted sperm extracts. Whole blots are presented. (E) Cells were fractionated into cytosol (Cyto) and membrane (Mem) fractions using Subcellular ProteoExtract kit, Calbiochem, according to the manufacturer’s instructions, and western blotted as described in ‘Materials and Methods’. PRKCA and p-(Thr497)-PRKCA were detected using specific antibodies. The results represent at least three independent experiments.

Article Snippet: Specific rabbit polyclonal antibody against phospho-PI3K p85 (Tyr458)/p55 (Tyr199) antibody and phospho-PRKA substrate (RRXS/T) (100G7) rabbit mAb were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Incubation, Western Blot, Membrane

Journal: REPRODUCTION

Article Title: Protein kinase A and protein kinase Cα/PPP1CC2 play opposing roles in the regulation of phosphatidylinositol 3-kinase activation in bovine sperm

doi: 10.1530/rep-09-0314

Figure Lengend Snippet: Figure 2 Activation of PI3K by cAMP/PRKA in sperm capacitation. Bovine sperm was incubated for 4 h in the capacitation medium (mTALP) (A–C) or in the bicarbonate-deficient medium (mTALP W/O HCOK 3) (A and C) in the presence or absence of (50 mM) H89 (A and C), (0.1 mM) 8Br-cAMP (cAMP) (A–C), and (162 nM) PMA (C). Sperm proteins were extracted at the indicated times and western blotted as described in ‘Materials and Methods’. PRKA-phospho-substrates (A) and p-PI3K (B and C) were detected using specific antibodies, and b-actin antibody was used to equalize the loading quantities. The results represent at least three independent experiments.

Article Snippet: Specific rabbit polyclonal antibody against phospho-PI3K p85 (Tyr458)/p55 (Tyr199) antibody and phospho-PRKA substrate (RRXS/T) (100G7) rabbit mAb were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Activation Assay, Incubation, Western Blot

Journal: REPRODUCTION

Article Title: Protein kinase A and protein kinase Cα/PPP1CC2 play opposing roles in the regulation of phosphatidylinositol 3-kinase activation in bovine sperm

doi: 10.1530/rep-09-0314

Figure Lengend Snippet: Figure 3 PRKC/PRKCA down-regulation enhances PI3K phosphorylation/activation. Bovine sperm was incubated for 4 h in mTALP in the presence or absence of PRKC inhibitors (0.1 nM) GF3200109X (GF) (A, C, and D) or (15 nM) Ro 32-0432 (Ro) (B–D) or with PRKA inhibitor (50 mM) H89 (D). Sperm proteins were extracted at the indicated times and western blotted as described in ‘Materials and Methods’. p-PI3K (A, B, and D) and PRKCA (C) were detected using specific antibodies, and b-actin or a-tubulin antibodies were used to equalize the loading quantities. (E) For detection of PI3K activity, sperm cells were incubated in mTALP with or without wort- mannin (WT; 10 nM), GF (0.1 nM) or H89 (50 mM) for 4 h. Sperm cells were stained with anti-PIP3(3,4,5), and the images were captured as described in ‘Materials and Methods’. The results represent at least five independent experiments.

Article Snippet: Specific rabbit polyclonal antibody against phospho-PI3K p85 (Tyr458)/p55 (Tyr199) antibody and phospho-PRKA substrate (RRXS/T) (100G7) rabbit mAb were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Phospho-proteomics, Activation Assay, Incubation, Western Blot, Activity Assay, Staining

Journal: REPRODUCTION

Article Title: Protein kinase A and protein kinase Cα/PPP1CC2 play opposing roles in the regulation of phosphatidylinositol 3-kinase activation in bovine sperm

doi: 10.1530/rep-09-0314

Figure Lengend Snippet: Figure 4 Serine/threonine phosphatase inhibitors induce time-dependent PI3K phosphorylation. Bovine sperm was incubated for 4 h in mTALP in the presence of calyculin-A (Caly; 100 nM) (A) or okadaic acid (OA; 1 mM) (B). (C) Bovine sperm was incubated for 4 h in mTALP in the presence or absence of calyculin-A (Caly) or okadaic acid (OA) at different concentrations as indicated. (D) Bovine sperm was incubated for 4 h in the capacitation medium (mTALP) or in the bicarbonate-deficient medium (mTALP W/O HCOK 3) in the presence or absence of (50 mM) H89 or (100 nM) calyculin-A (Caly). Sperm proteins were extracted at the indicated times and western blotted as described in ‘Materials and Methods’. p-PI3K (A–D), p-(Ser657)-PRKCA (A and B), and PRKCA (A and B) were detected using specific antibodies, and b-actin (A, B, and D) or tubulin (C) antibodies were used to equalize the loading quantities. The results represent at least three independent experiments.

Article Snippet: Specific rabbit polyclonal antibody against phospho-PI3K p85 (Tyr458)/p55 (Tyr199) antibody and phospho-PRKA substrate (RRXS/T) (100G7) rabbit mAb were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Phospho-proteomics, Incubation, Western Blot

Journal: REPRODUCTION

Article Title: Protein kinase A and protein kinase Cα/PPP1CC2 play opposing roles in the regulation of phosphatidylinositol 3-kinase activation in bovine sperm

doi: 10.1530/rep-09-0314

Figure Lengend Snippet: Figure 5 PMA-induced PRKCA degradation enhances PI3K phosphorylation/activation. (A) Bovine sperm was incubated for 7 h in mTALP in the presence or absence of PRKC activator PMA (162 nM). Sperm proteins were extracted at the indicated times and western blotted as described in ‘Materials and Methods’. p-PI3K, p-(Ser657)-PRKCA, and PRKCA were detected using specific antibodies, and b-actin antibody was used to equalize the loading quantities. The result represents at least four independent experiments. (B) Bovine sperm was incubated for 10 min in mTALP in the presence or absence of PMA (162 nM). At the indicated times, cells were fractionated into cytosol (Cyto) and membrane (Mem) fractions using Subcellular ProteoExtract kit, Calbiochem, according to the manufacturer’s instructions, and western blotted as described in ‘Materials and Methods’. PRKCA was detected using specific antibody. The result represents two independent experiments. (C) Bovine sperm was incubated for 30 min in mTALP in the presence or absence of 10 mM calcium ionophore A23187 (AR inducer). Sperm proteins were extracted at the indicated times and western blotted as described in ‘Materials and Methods’. PRKCA was detected using specific antibody, and tubulin antibody was used to equalize the loading quantities. The result represents three independent experiments.

Article Snippet: Specific rabbit polyclonal antibody against phospho-PI3K p85 (Tyr458)/p55 (Tyr199) antibody and phospho-PRKA substrate (RRXS/T) (100G7) rabbit mAb were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Phospho-proteomics, Activation Assay, Incubation, Western Blot, Membrane

Journal: REPRODUCTION

Article Title: Protein kinase A and protein kinase Cα/PPP1CC2 play opposing roles in the regulation of phosphatidylinositol 3-kinase activation in bovine sperm

doi: 10.1530/rep-09-0314

Figure Lengend Snippet: Figure 8 Diagrammatic representation of the mechanisms underlying PRKA-mediated PI3K activation during bovine sperm capacitation. At the beginning of the capacitation process, PRKCA and PPP1CC2 are active and present in high levels and inhibit (directly or indirectly) PI3K phosphorylation. During the capacitation, PRKA is activated by cAMP that is generated by the bicarbonate-activated soluble adenylyl cyclase (sAC). PRKA phosphorylates PI3K leading to its activation. PI3K activation cannot be seen at beginning of the capacitation, because the PRKCA-activated PPP1CC2 removes PI3K phosphorylation. With the ongoing capacitation, PRKA mediates PRKCA and PPP1CC2 degradation, which can be proteasome dependent. Therefore, PI3K inhibition by PRKCA/PPP1CC2 is removed, and PI3K phosphorylation/activation by PRKA can occur.

Article Snippet: Specific rabbit polyclonal antibody against phospho-PI3K p85 (Tyr458)/p55 (Tyr199) antibody and phospho-PRKA substrate (RRXS/T) (100G7) rabbit mAb were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Activation Assay, Phospho-proteomics, Generated, Inhibition