Fig 1: AKT inhibition suppresses Agrin-induced AChR cluster formation in C2C12 myotubes. (A) Immunoblot analysis showing reduced phosphorylation of AKT (pSer474) and its downstream targets S6 (pSer240/244) and PRAS40 (pThr240 following MK2206 treatment (10 μM) for 3 h or 6 h. (B) Western blot analysis of PAN-AKT, pSer240/244-S6, S6 and HSP90 in C2C12 myotubes treated with MK2206 (10 μM, 3 h or 6 h) following Agrin treatment for 16 h. (C) Representative images of AChR cluster intensity visualized by fluorescent α-bungarotoxin (α-bungarotoxin) staining in C2C12 myotubes treated with n-Agrin, with or without MK2206 pretreatment. MK2206 markedly reduced the intensity of Agrin-induced AChR clusters. (D) mRNA expression of AChR subunits (α, δ, ε) in C2C12 myotubes treated with neural Agrin (n-Agrin, 16 h) with or without MK2206 pretreatment. MK2206 significantly reduced Agrin-induced expression of AChRα and AChRδ and robustly suppressed AChRε expression. Data are presented as mean ± SEM; * p < 0.05, *** p < 0.001, **** p < 0.0001. Images were acquired using a 40× objective; scale bar, 20 µm.
Fig 2: AKT regulates NMJ-associated gene transcription during both formation and maintenance of AChR clusters. (A) qPCR analysis of NMJ-associated genes (LRP4, MuSK, Agrin, and Utrophin) during the formation phase. (B) qPCR analysis of NMJ-associated genes during the formation and maintenance phase. mRNA expression was normalized to the housekeeping gene (TBP-1) and expressed as fold change relative to control. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. unstimulated control or Agrin treatment.
Fig 3: AKT is required for the maintenance of Agrin-induced AChR cluster intensity. (A) Confocal images of AChR clusters intensity visualized with Alexa547-conjugated α-bungarotoxin in C2C12 myotubes treated with Agrin alone for 16 h or Agrin + MK2206 for 3 h or 6 h. (B) qPCR analysis of AChR subunit transcripts (α, δ, and ε) after 3 h or 6 h of MK2206 treatment during the final phase of Agrin stimulation for 16 h. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. unstimulated control. Images were acquired using a 40× objective; scale bar, 20 µm.
Fig 4: AKT maintains Agrin-mediated AChR clusters via the proteasome degradation pathway. (A) Representative confocal images of AChR clusters labeled with α-bungarotoxin. MK2206 treatment (6 h) markedly reduced AChR cluster intensity, whereas pretreatment with the proteasome inhibitor MG132 (10 μM, 30 min) restored cluster abundance. (B) qPCR analysis of AChR transcripts in C2C12 myotubes treated with Agrin or Agrin ± MK2206 and MG132. (C) qPCR analysis of proteasome-related genes (Psma1, Psmc4, and Psmd11) in C2C12 myotubes treated with Agrin ± MK2206 or Agrin + MK2206 and MG132. Data are presented as mean ± SEM. * p < 0.05 vs. unstimulated control, ** p < 0.01 vs. unstimulated Agrin, *** p < 0.001 vs. Agrin+MK2206. **** p < 0.0001. Images were acquired using a 40× objective. Images were acquired using a 40× objective; scale bar, 20 µm.
Supplier Page from MedChemExpress for Agrin Protein, Human (CHO, His)