Journal: Journal of Cellular and Molecular Medicine

Article Title: Phosphorylation‐mediated PI3K‐Art signalling pathway as a therapeutic mechanism in the hydrogen‐induced alleviation of brain injury in septic mice

doi: 10.1111/jcmm.17568

Figure Lengend Snippet: Differentially expressed phosphoproteins (DPPs) (Rps6, Pten, Ywhag, mTOR, Magi1, and Pkn2) participate in the PI3K‐AKT signalling pathway were significantly different in the therapeutic mechanism of hydrogen in CLP‐induced mice. (A) The western blot were used to identify the differentially expressed phosphoproteins and the total proteins. The results showed there were no changes in the total level of Ywhag, mTOR, and Magi1 quantified by the ratio of band density to those of β‐actin, respectively (B) and the MS/MS spectrums from Phosphorylated peptides. (C) (p‐Ywhag assigned to TSADGNEKK; p‐Magi1 assigned to AENEVPSPASSHHSSNQPASLTEEK; p‐mTOR assigned to IMLRMAPDYDHLTLMQKVEVFEHAVNNTAGDDLAK). The b and y ions including loss of ammonia and water were considered when we calculated the PTM score.) (D) The expression of Rps6, p‐Rps6, Pten, p‐Pten, mTOR, p‐mTOR, Pkn2, and p‐Pkn2 in CLP + H2 were detected by western blot. Quantitative analysis of p‐Rps6, p‐Pten, p‐mTOR, and p‐Pkn2 is shown as the ratio of band density to that of Rps6, Pten, mTOR, and Pkn2, respectively. NS, no significance; * p < 0.05; ** p < 0.01; *** p < 0.001. Magi1, membrane‐associated guanylate kinase1; mTOR, mammalian target of rapamycin; Pkn2, protein kinase N2; Pten, phosphatase and tensin homologue deleted on chromosome ten; Rps6, Ribosomal protein S6; Ywhag/14–3‐3, tyrosine 3‐Monooxygenase/tryptophan 5‐monooxygenase activation protein gamma

Article Snippet: The membrane was blocked using 5% skim milk powder in Tris/Tween‐20 for 2 h and then incubated with the following primary antibodies at 4°C overnight: Rps6 (1:1000, cat# ab225676, Abcam), Ywhag/14‐3‐3 (1:500, cat# DF3493, Affinity), PTEN (1:1000, cat# ab267787, Abcam), Magi1 (1:500, cat# 55048‐1‐AP, Proteintech), mTOR (1:10000, cat# ab134903, Abcam), Pkn2 (1:1000, cat# ab138514, Abcam), phosphorylated Rps6 (S240 + S244) (1:5000, cat# ab215214, Abcam), phosphorylated PTEN (1:500, cat# DF3493, Affinity), phosphorylated mTOR (S2448) (1:1000, cat# ab109268, Abcam), phosphorylated Pkn2 (T816) (1:1000, cat# ab124709, Abcam), and β‐actin (1: 5000, Proteintech).

Techniques: Western Blot, Tandem Mass Spectroscopy, Expressing, Membrane, Activation Assay