Fig. 7.

Macrophage SAMSN1 activates AMPKα2 through GAB1/SHP2/PKA pathway. (A–B) BMDMs were isolated from Samsn1^MKO, Samsn1^MTG or control mice and stimulated with LPS (100 ng/mL) for 12 h, and then intracellular cAMP level and PKA activity were detected. (C) BMDMs from Samsn1^WT or Samsn1^MTG mice were incubated with RpAMPS (10 μmol/L) or H89 (10 μmol/L) for 24 h and stimulated with LPS (100 ng/mL) for an additional 12 h, and then AMPKα (T172) phosphorylation was detected. (D) BMDMs from Samsn1^WT or Samsn1^MTG mice were transfected with siEpac or siScr at a dose of 50 nmol/L using Lipofectamine® RNAiMAX Transfection Reagent for 6 h, and then cultured in fresh DMEM medium for 48 h, followed by the stimulation with LPS (100 ng/mL) for an additional 12 h. AMPKα (T172) phosphorylation was detected. (E) BMDMs were transfected with siEpac or siScr at a dose of 50 nmol/L using Lipofectamine® RNAiMAX Transfection Reagent for 6 h, and then cultured in fresh DMEM medium for 48 h, followed by the stimulation with LPS (100 ng/mL) for an additional 12 h. Epac mRNA level was detected. (F) BMDMs from Samsn1^WT or Samsn1^MTG mice were stimulated with LPS (100 ng/mL) for 12 h, and then Gab1 mRNA level was detected. (G) BMDMs were lysed and exposed to IP assay to detect endogenous interaction between SAMSN1 and GAB1. (H–I) BMDMs from Samsn1^WT or Samsn1^MTG mice were stimulated with LPS (100 ng/mL) for 12 h, and then GAB1 protein level was detected. (J) BMDMs from Samsn1^WT or Samsn1^MTG mice were incubated with CHX (10 μmol/L) for indicating times in the presence of LPS (100 ng/mL), and then GAB1 protein level was detected. (K) BMDMs were transfected with siGab1 or siScr at a dose of 50 nmol/L using Lipofectamine® RNAiMAX Transfection Reagent for 6 h, and then cultured in fresh DMEM medium for 48 h, followed by the stimulation with LPS (100 ng/mL) for an additional 12 h Gab1 mRNA level was detected. (L) BMDMs from Samsn1^WT or Samsn1^MTG mice were transfected with siGab1 (50 nmol/L) or infected with lentiviral vectors carrying GAB1Y627F or SHP2 DN (MOI = 20) for 6 h, and then cultured in fresh DMEM medium for an additional 48 h, followed by the stimulation with LPS (100 ng/mL) for an additional 12 h. PKA activity was detected. (M) The levels of TNF-α and IL-6 in the medium were detected. (N) The level of ROS was detected. n = 6 per group, differences with P value < 0.05 were considered statistically significant. NS indicates no statistically significant.