anti pakt  (Proteintech)

Journal: Animal Nutrition

Article Title: Proline promotes prolyl 4-hydroxylase subunit alpha 1 via transforming growth factor beta 1 to increase the synthesis and deposition of collagen in grass carp ( Ctenopharyngodon idellus ) muscle fibroblasts: Insights from integrated bioinformatics and functional analysis

doi: 10.1016/j.aninu.2025.06.014

Figure Lengend Snippet: Relative expression of transforming growth factor-β (TGFβ)/SMADs signaling pathway-related genes and proteins treated with proline (Pro) for 48 h. (A) mRNA relative expression of tgfb1 . (B) mRNA relative expression of smad2 . (C) mRNA relative expression of smad3 . (D) mRNA relative expression of smad4 . (E) Effects of Pro on the protein levels of SMAD2, p-SMAD2 (Ser250), SMAD3, and p-SMAD3 (Ser423/Ser425). (F) Western blot analysis of SMAD2. Histogram represents the ratio between SMAD2 and GAPDH. (G) Western blot analysis of the phosphorylation level of SMAD2. Histogram represents the ratio between the phosphorylated protein and the total amount of SMAD2. (I) Western blot analysis of SMAD3. Histogram represents the ratio between SMAD3 and GAPDH. (J) Western blot analysis of the phosphorylation level of SMAD3. Histogram represents the ratio between the phosphorylated protein and the total amount of SMAD3. Data represents mean and standard error of the mean (SEM) ( n = 3). Significant differences between groups are represented by different letters ( P < 0.05). Abbreviations and full names of the genes and proteins are provided in Table S1.

Article Snippet: Primary antibodies included SMAD2 antibody (1: 1000), phospho-SMAD2 antibody (Ser250) (1: 1000), SMAD3 antibody (1: 1000), phospho-SMAD3 antibody (Ser423/Ser425) (1: 1000) and GAPDH antibody (1: 5000) which were purchased from MedChemExpress LLC (Monmouth Junction, NJ, USA).

Techniques: Expressing, Western Blot, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: A new functional assay reveals that membrane binding is critical for overactivation of the phosphoinositide 3-kinase H1047R mutant

doi: 10.1016/j.jbc.2026.111207

Figure Lengend Snippet: Establishment and titration of the optimal form of the substrate PIP 2 . A , the optimal form of substrate was determined by comparing two different ratios of lPIP 2 to total lipids, 1:10 and 1:2, both at a final lPIP 2 concentration of 7 μM. A water-soluble PIP 2 was also tested, at a concentration of 100 μM. Note that the most advantageous substrate preparation is a 1:2 mixture of lPIP 2 to total lipids. Two replicates from n = 10. B , different concentrations of lPIP 2 (1:2 mixture of lPIP 2 to total lipids) were tested. Note the dose-dependent effect. Two replicates from n = 4. C , schematic diagram of the experimental set up used in ( A ) and ( B ). The individual data points displayed above represent the means of replicates in each experiment. The statistical significance of difference between groups was examined by one-way ANOVA, followed by Tukey’s multiple comparisons test. ns : no significance, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Data were shown as mean ± SD. Specific activity values for PI3Kα, calculated as pmol PIP 3 produced per minute per nanogram of enzyme, are indicated in red fonts in the figure. lPIP 2 , lipid form of PIP 2 ; PIP 2 , phosphatidylinositol-4,5-biphospate; PIP 3 , phosphatidylinositol-3,4,5-triphosphate.

Article Snippet: Lipid PIP 2 [L-α-phosphatidylinositol-4,5 bisphosphate (Brain, Porcine) (ammonium salt)] and soluble PIP 2 [1,2-dioctanoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5-bisphosphate) (ammonium salt)] were from Avanti Polar Lipids.

Techniques: Titration, Concentration Assay, Activity Assay, Produced

Journal: The Journal of Biological Chemistry

Article Title: A new functional assay reveals that membrane binding is critical for overactivation of the phosphoinositide 3-kinase H1047R mutant

doi: 10.1016/j.jbc.2026.111207

Figure Lengend Snippet: Role of membranes in the overactivation of the mutants H1047R and E545K. We compare the PI3Kα enzymatic activity of H1047R, E545K, and WT, using as substrate 7 μM lPIP 2 ( A ), or 100 μM sPIP 2 ( B ). The activities of the mutants were normalized to WT, which was set to value “1.” Note that E545K shows higher activity than WT with either soluble or membranous PIP 2 , whereas H1047R exhibits increased activity only when membranes are used. Two replicates from n = 6 in ( A ) and n = 7 in ( B ). The individual data points displayed above represent the means of replicates in each experiment. The statistical significance of difference between groups in graphs ( A ) and ( B ) was examined by one-way ANOVA, followed by Tukey’s multiple comparisons test. ns : no significance, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Data were shown as mean ± SD. Specific activity values for PI3Kα, calculated as pmol PIP 3 produced per minute per nanogram of enzyme, are indicated in red fonts in the figure. lPIP 2 , lipid form of phosphatidylinositol-4,5-biphospate; SPR, surface plasmon resonance; sPIP 2 , soluble form of PIP 2 ; PIP 3 , phosphatidylinositol-3,4,5-triphosphate.

Article Snippet: Lipid PIP 2 [L-α-phosphatidylinositol-4,5 bisphosphate (Brain, Porcine) (ammonium salt)] and soluble PIP 2 [1,2-dioctanoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5-bisphosphate) (ammonium salt)] were from Avanti Polar Lipids.

Techniques: Activity Assay, Produced, SPR Assay

Journal: Animal Nutrition

Article Title: Proline promotes prolyl 4-hydroxylase subunit alpha 1 via transforming growth factor beta 1 to increase the synthesis and deposition of collagen in grass carp ( Ctenopharyngodon idellus ) muscle fibroblasts: Insights from integrated bioinformatics and functional analysis

doi: 10.1016/j.aninu.2025.06.014

Figure Lengend Snippet: Relative expression of transforming growth factor-β (TGFβ)/SMADs signaling pathway-related genes and proteins treated with proline (Pro) for 48 h. (A) mRNA relative expression of tgfb1 . (B) mRNA relative expression of smad2 . (C) mRNA relative expression of smad3 . (D) mRNA relative expression of smad4 . (E) Effects of Pro on the protein levels of SMAD2, p-SMAD2 (Ser250), SMAD3, and p-SMAD3 (Ser423/Ser425). (F) Western blot analysis of SMAD2. Histogram represents the ratio between SMAD2 and GAPDH. (G) Western blot analysis of the phosphorylation level of SMAD2. Histogram represents the ratio between the phosphorylated protein and the total amount of SMAD2. (I) Western blot analysis of SMAD3. Histogram represents the ratio between SMAD3 and GAPDH. (J) Western blot analysis of the phosphorylation level of SMAD3. Histogram represents the ratio between the phosphorylated protein and the total amount of SMAD3. Data represents mean and standard error of the mean (SEM) ( n = 3). Significant differences between groups are represented by different letters ( P < 0.05). Abbreviations and full names of the genes and proteins are provided in Table S1.

Article Snippet: Primary antibodies included SMAD2 antibody (1: 1000), phospho-SMAD2 antibody (Ser250) (1: 1000), SMAD3 antibody (1: 1000), phospho-SMAD3 antibody (Ser423/Ser425) (1: 1000) and GAPDH antibody (1: 5000) which were purchased from MedChemExpress LLC (Monmouth Junction, NJ, USA).

Techniques: Expressing, Western Blot, Phospho-proteomics