Fig. 7

NCAM1 assists αV/ β1 in tau PFF-induced astrogliosis. (A) Conditioned medium from wild type or Ncam1 knockdown astrocytes treated with the indicated agents was analyzed by immunoblotting. The lower panel shows the protein level of NCAM1 in the corresponding cell lysates. The gels represent one of the three biological repeats. (B) The graphs show the quantification of C3 and MMP3 secretion in tau PFF-treated control and NCAM1 knockdown cells in three independent experiments. **, p < 0.01, ***, p < 0.001 by unpaired Student’s t-test. n = 3 biological repeats. (C, D) qRT-PCR analysis of the indicated cytokine (C) and chemokine genes (D) in wild type PAs or in PAs with the indicated gene knockdown after treatment with tau PFF. The graphs show Log Fold Change normalized to PBS-treated cells. Error bars, means ± SD. (E, F) Representative gels show that Tau PFF-induced phosphorylation of AKT (pAKT) requires NCAM1. Wild type PAs or Ncam1 knockdown astrocytes were treated with tau PFF or SC79 as a control. Cell lysates were analyzed by immunoblotting (E). Note that Ncam1 knockdown reduces total AKT and GFAP possibly because of a growth defect associated with Ncam1 knockdown [55]. pAKT normalized to total AKT was quantified in (F). Error bars, means ± SD. ***, p < 0.001 by unpaired student’s t-test. n = 3 biological repeats. (G) Co-immunoprecipitation shows a physical interaction between tau PFF and endogenous NCAM1. Astrocytes were treated with tau PFF or PBS as a control. Cell lysates were subjected to immunoprecipitation with either a control IgG or a tau specific antibody (tau-46). Cell lysates and precipitated proteins were analyzed by immunoblotting. Asterisk indicates a non-specific band. Shown are blots representing 3 biological repeats