Fig. 7

Impairment of p65 NF-κB nuclear translocation in Atg7 deficient microglia upon LPS treatment. Microglial cells, i.e. shAtg7 and shCtrl BV2 cells were treated with LPS (100 ng/ml) for 15 min or 30 min. a Comparison of Nfkb1, Nfkb2, Rel, Rel and Relb (NF-κB family members) mRNA expression measured by RT-qPCR in shAtg7 and shCtrl BV2 cells. b Immunoblot analysis of p65 NF-κB catalytic subunit in cytoplasmic versus nuclear subcellular fractions in shAtg7 and shCtrl BV2 cells. The expression of LAMIN C and GAPDH were used as loading controls for cytoplasmic and nuclear fraction, respectively. c Graphs show the quantification for p65 NF-κB protein expression in cytoplasm and nucleus in untreated shAtg7 and shCtrl BV2 cells (total expression in both subcellular fractions set to 1, for shCtrl BV2 cells). d Graphs show the quantification of the percentual localization of total p65 NF-κB protein expression in cytoplasmic versus nuclear fractions in shAtg7 and shCtrl BV2 cells under each condition, i.e. untreated, 15 min or 30 min LPS-treatment. e ChIP analysis of p65 NF-κB enrichment at a Nos2 promoter region encompassing a kB DNA binding site in shAtg7 and shCtrl BV2 cells treated with LPS (100 ng/ml) for 1 h. IgG was used as control. Values are a mean of 3 (e) 4 (a), or 5 (c, d) independent experiments ± SEM and considered significant for *p < 0.05, **p < 0.01, ***p < 0.001, ****p > 0.0001. n.s., not significant for the indicated comparison