rabbit anti p akt  (Bioss)

Journal: Acta Physiologica (Oxford, England)

Article Title: NMDA‐Type Glutamate Receptor Activation Promotes Ischemic Arrhythmias by Targeting the AKT1–TBX3–Nav1.5 Axis

doi: 10.1111/apha.70085

Figure Lengend Snippet: AKT1 is the predominant AKT isoform in myocardium and post‐translationally upregulates TBX3. (a) Protein levels of TBX3 and Nav1.5 in H9C2 cells treated with MK2206 at different time points. (b, c) mRNA levels of Nav1.5 and TBX3 in H9C2 cells treated with MK2206 at different time points. (d) Bioinformatics analysis based on the reported single‐cell transcriptome data. The original dataset is GSE126128 . (e) qRT‐PCR analyses using primers specific to AKT1, AKT2, and AKT3 of human, mice, and H9C2 cells. (f) Screening of the best small hairpin RNAs for AKT1 (sh‐AKT1) using Western blot. (g) AKT1 knockdown caused a decrease in TBX3 protein level and an increase in Nav1.5 protein level. Data from three independent repetitions of experiments are expressed as means ± standard deviations (SDs). Two‐tailed Student's t test was performed for comparison between two groups, and one‐way ANOVA followed by a post hoc Tukey's test was used to compare the data from multiple groups. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti‐NR1 (1:1000, Abcam), rabbit polyclonal anti‐p‐NR1 (1:1000, Abcam), rabbit polyclonal anti‐Nav1.5 (1:1000, Proteintech), rabbit monoclonal anti‐Kv11.1 (1:1000, Abcam), rabbit polyclonal anti‐Kv4.2 (1:1000, Proteintech), mouse monoclonal anti‐Kv7.1 (1:1000, Abcam), rabbit polyclonal anti‐Kir2.1 (1:1000, Proteintech), rabbit polyclonal anti‐Cav1.2 (1:1000, Proteintech), rabbit polyclonal anti‐TBX3 (1:1000, Abcam), mouse monoclonal anti‐Flag M2 (1:1000, Sigma‐Aldrich), rabbit polyclonal AKT (1:1000, Cell Signaling), rabbit polyclonal p‐AKT (1:1000, Cell Signaling), rabbit polyclonal AKT1 (1:1000, Cell Signaling), rabbit polyclonal p‐AKT1 (1:1000, Cell Signaling), mouse polyclonal anti‐tubulin (1:1000, Proteintech), and mouse polyclonal anti‐actin (1:1000, Proteintech).

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Two Tailed Test, Comparison

Journal: Acta Physiologica (Oxford, England)

Article Title: NMDA‐Type Glutamate Receptor Activation Promotes Ischemic Arrhythmias by Targeting the AKT1–TBX3–Nav1.5 Axis

doi: 10.1111/apha.70085

Figure Lengend Snippet: AKT1 promotes TBX3 protein stability and enhances its ability to repress Nav1.5 by pseudo‐phosphorylation of TBX3 at serine 720. (a) MG132, an inhibitor of the proteasome, rescued TBX3 protein levels in AKT1‐silenced H9C2 cells. (b, c) The decay rates of TBX3 protein level in both H9C2 cells and AKT1‐silenced H9C2 cells were evaluated using Western blot. (d, e) AKT1 phosphorylation of TBX3 at serine 720 enhanced the ability to inhibit Nav1.5 in H9C2 cells, identified using Western blot and RT‐PCR. Data from three independent repetitions of experiments are expressed as means ± standard deviations (SDs). One‐way ANOVA followed by a post hoc Tukey's test was used to compare the data from multiple groups. (** p < 0.01, *** p < 0.001).

Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti‐NR1 (1:1000, Abcam), rabbit polyclonal anti‐p‐NR1 (1:1000, Abcam), rabbit polyclonal anti‐Nav1.5 (1:1000, Proteintech), rabbit monoclonal anti‐Kv11.1 (1:1000, Abcam), rabbit polyclonal anti‐Kv4.2 (1:1000, Proteintech), mouse monoclonal anti‐Kv7.1 (1:1000, Abcam), rabbit polyclonal anti‐Kir2.1 (1:1000, Proteintech), rabbit polyclonal anti‐Cav1.2 (1:1000, Proteintech), rabbit polyclonal anti‐TBX3 (1:1000, Abcam), mouse monoclonal anti‐Flag M2 (1:1000, Sigma‐Aldrich), rabbit polyclonal AKT (1:1000, Cell Signaling), rabbit polyclonal p‐AKT (1:1000, Cell Signaling), rabbit polyclonal AKT1 (1:1000, Cell Signaling), rabbit polyclonal p‐AKT1 (1:1000, Cell Signaling), mouse polyclonal anti‐tubulin (1:1000, Proteintech), and mouse polyclonal anti‐actin (1:1000, Proteintech).

Techniques: Phospho-proteomics, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Acta Physiologica (Oxford, England)

Article Title: NMDA‐Type Glutamate Receptor Activation Promotes Ischemic Arrhythmias by Targeting the AKT1–TBX3–Nav1.5 Axis

doi: 10.1111/apha.70085

Figure Lengend Snippet: NMDAR activation promotes post‐MI arrhythmias through the AKT1–TBX3–Nav1.5 axis. (a) Protein levels of p‐AKT1 and quantitative analysis in patients with ischemic heart disease evaluated using Western blot. (b) Protein levels of p‐AKT1 and quantitative analysis in MI mice evaluated using Western blot. (c) Protein levels of p‐AKT1 and quantitative analysis in H9C2 cells evaluated using Western blot. (d) NMDAR activation failed to increase TBX3 expression and decrease Nav1.5 levels when inhibiting AKT1 phosphorylation. (e) NMDAR activation failed to promote the expression of TBX3 and decrease Nav1.5 levels in AKT1‐silenced H9C2 cells. Data from three independent repetitions of experiments are expressed as means ± standard deviations (SDs). Two‐tailed Student's t test was performed for comparison between two groups, and one‐way ANOVA followed by a post hoc Tukey's test was used to compare the data from multiple groups. * p < 0.05, ** p < 0.01.

Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti‐NR1 (1:1000, Abcam), rabbit polyclonal anti‐p‐NR1 (1:1000, Abcam), rabbit polyclonal anti‐Nav1.5 (1:1000, Proteintech), rabbit monoclonal anti‐Kv11.1 (1:1000, Abcam), rabbit polyclonal anti‐Kv4.2 (1:1000, Proteintech), mouse monoclonal anti‐Kv7.1 (1:1000, Abcam), rabbit polyclonal anti‐Kir2.1 (1:1000, Proteintech), rabbit polyclonal anti‐Cav1.2 (1:1000, Proteintech), rabbit polyclonal anti‐TBX3 (1:1000, Abcam), mouse monoclonal anti‐Flag M2 (1:1000, Sigma‐Aldrich), rabbit polyclonal AKT (1:1000, Cell Signaling), rabbit polyclonal p‐AKT (1:1000, Cell Signaling), rabbit polyclonal AKT1 (1:1000, Cell Signaling), rabbit polyclonal p‐AKT1 (1:1000, Cell Signaling), mouse polyclonal anti‐tubulin (1:1000, Proteintech), and mouse polyclonal anti‐actin (1:1000, Proteintech).

Techniques: Activation Assay, Western Blot, Expressing, Phospho-proteomics, Two Tailed Test, Comparison

Journal: Molecular Vision

Article Title: Astragaloside IV improves the survival rates of retinal ganglion cells in traumatic optic neuropathy by regulating autophagy mediated by the AMPK-MTOR-ULK signaling pathway

doi:

Figure Lengend Snippet: AS-IV enhances autophagy by regulating the phosphorylation of AMPK, mTOR, and ULK. A . Western blot was used to assess the AMPK, mTOR, and ULK phosphorylation levels in retinal tissue of rats in all groups ( n = 3). * p < 0.05 or ** p < 0.01, compared with the MO group (excluding the CC group). # p < 0.05 or ## p < 0.01, comparison between all drug groups. B . LC3 protein levels in retinal tissue ( n = 3). * p < 0.05, comparison between AS-IV and 3-MA group. C . The AKT1 and S6RP phosphorylation levels in retinal tissue ( n = 3); * p < 0.05, comparison between AS-IV and MO group.

Article Snippet: The following materials were purchased for this study: AS-IV ( B20564 ; Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China), rapamycin (ab146591; Abcam, Cambridge, UK), compound C (dorsomorphin, ab120843; Abcam), LC3B (E5Q2K) mouse mAb (83506S; CST, Boston, MA), AMPKα (D63G4) rabbit mAb (40308ES20; CST), mTOR (7C10) rabbit mAb (2983S; CST), ULK1 (D8H5) rabbit mAb (8054S; CST), anti-phospho-AMPKα (Thr172) rabbit mAb (50081S; CST), phospho-mTOR (Ser2448; D9C2) XP rabbit mAb (5536S; CST), phospho-ULK1 (Ser555; D7O6U) rabbit mAb (14202S; CST), anti-Brn3A antibody (ab245230; Abcam), rabbit anti-phospho-RPS6 (Ser240 + Ser244) polyclonal antibody (bs-3389R; Bioss, Beijing, China), rabbit anti-phospho-AKT1 (Ser473) polyclonal antibody (bs-12456R; Bioss), AKT monoclonal antibody (bsm-52010R; Bioss), 3-MA (HY-19312; Monmouth Junction, NJ), TUNEL Apoptosis Detection Kit (40308ES20; YEASEN, Shanghai, China), goat anti-mouse IgG-HRP (ab6789; Abcam), and goat anti-rabbit IgG-HRP (ab205718; Abcam).

Techniques: Phospho-proteomics, Western Blot, Comparison