Journal: Brain Communications

Article Title: Gonadotropin-releasing hormone alleviates chronic pain-related depression in male mice by rebalancing the anterior cingulate cortex excitatory-inhibitory processes via the protein kinase C/Erb-B2 receptor tyrosine kinase 4 pathway

doi: 10.1093/braincomms/fcag138

Figure Lengend Snippet: GnRH activates ErbB4 via the PKC pathway in the ACC of mice after CFA injection . ( A ) Effects of Trip treatment on the protein levels of PKCα and p-MARCKS are demonstrated by western blot and the semiquantitative analysis of PKCα and p-MARCKS relative to β-actin and MARCKS, and the original uncropped blots are shown in . ( B ) The experimental timeline for Trip and bisindolylmaleimide I treatment after CFA injection. ( C ) Effects of Trip and bisindolylmaleimide I treatment on the total movement distance by mice in the OFT. ( D ) Effects of Trip and bisindolylmaleimide I treatment on the sucrose preference rate of 1% sucrose solution in the SCPT. ( E ) Effects of Trip and bisindolylmaleimide I treatment on the immobility time within 4 min in the TST. ( F ) Effects of Trip and bisindolylmaleimide I treatment on the immobility time within 4 min in the FST. ( G ) Effects of Trip and bisindolylmaleimide I treatment on the protein levels of PKCα, p-MARCKS and p-ErbB4 were demonstrated by western blot and the semiquantitative analysis of these proteins relative to β-actin, MARCKS and ErbB4, and the original uncropped blots are shown in . ( H ) Effects of Trip and bisindolylmaleimide I treatment on the protein levels of GAD67, VGAT, vGluT1 and vGluT2 demonstrated by western blot and the semiquantitative analysis of these proteins relative to β-actin, and the original uncropped blots are shown in . n = 6 mice for western blot test and n = 6–9 mice per group for behaviour tests and any value >2 SD from the group mean was considered an outlier and excluded from the analysis. One-way ANOVAs followed by Bonferroni’s post hoc testing for multiple comparisons among the groups. Each data point represents the data of one mouse. * P < 0.05, ** P < 0.01 and *** P < 0.001. Abbreviations: ACC, anterior cingulate cortex; NS, normal saline; CFA, complete Freund’s adjuvant; ErbB4, Erb-B2 receptor tyrosine kinase 4; p-ErbB4, phosphorylated-Erb-B2 receptor tyrosine kinase 4; GnRH, gonadotropin-releasing hormone; GnRHR, gonadotropin-releasing hormone receptor; PKCα, protein kinase C α; MARCKS, myristoylated alanine-rich C kinase substrate; p-MARCKS, phosphorylated-myristoylated alanine-rich C kinase substrate; Trip, triptorelin; Bis I, bisindolylmaleimide I; GAD67, glutamate decarboxylase 67; VGAT, vesicular γ-aminobutyric acid transporter; vGluT1, vesicular glutamate transporter 1; vGluT2, vesicular glutamate transporter 2; OFT, open field test; SCPT, sucrose preference test; TST, tail suspension test; FST, forced swimming test.

Article Snippet: GnRH antagonist, cetrorelix (CEX, 0.1 mg/kg, Meilunbio); ErbB4 inhibitor, dacomitinib (1 mg/kg, Targetmol); or PKC inhibitor, bisindolylmaleimide I (0.02 mg/kg, Targetmol) were dissolved in NS and injected intraperitoneally on Days 19 through 21 after CFA or NS injection.

Techniques: Injection, Western Blot, Saline, Adjuvant, Suspension

Journal: The Journal of Biological Chemistry

Article Title: C-spine mutations of protein kinase C and Akt as a novel generalizable approach to create stable pseudokinases

doi: 10.1016/j.jbc.2025.110921

Figure Lengend Snippet: Akt1 C-spine mutants are inactive with reduced phosphorylation at the hydrophobic motif. A – B , Akt activity in COS7 cells expressing BKAR and empty vector ( black trace ) or coexpressing BKAR and mCherry-Akt1 WT, V164F, or A177F. Cells grown in 10% serum A or serum starved for 18 h before imaging B . At 3 min, cells were treated with A , 20 μM of the Akt inhibitor, GDC0068 alone, or B , 50 ng/ml EGF followed by 20 μM of GDC0068 at 10 min. Changes in FRET/CFP ratios were normalized to the first 3 min A and plotted or absolute changes in FRET/CFP ratios were plotted B . Data are representative of 36 to 46 cells per condition from three to four independent experiments (mean ± SEM). C , representative western blot of whole cell lysate from COS7 cells transfected with the indicated HA-tagged Akt1 constructs. Cells were serum starved for 15 h before treating with DMSO or 50 ng/ml EGF for 8 min. D – F , quantification of Akt1 at the activation loop D , turn motif E , and hydrophobic motif F , from panel C normalized to loading control and total Akt1 protein. Data were normalized to the phosphorylation of DMSO-treated Akt1 WT and represent mean ± SEM from six independent experiments. ns = nonsignificant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 by two-way ANOVA and Tukey post hoc test. Relevant statistical comparisons are shown. Akt, protein kinase B; BKAR, B kinase activity reporter; CFP, cyan fluorescent protein; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; HA, hemagglutinin.

Article Snippet: Compound 20 (C20) (cat. no. S6577) and GDC0068 (cat. no. S2808) were purchased from Selleckchem.

Techniques: Phospho-proteomics, Activity Assay, Expressing, Plasmid Preparation, Imaging, Western Blot, Transfection, Construct, Activation Assay, Control