Fig. 5.

GSK3β phosphorylation of PELP1 results in reduced stability of PELP1. (A) Tissue sections derived from sham, placebo, and E2-treated mice at 24 h after GCI were blocked in 5% (vol/vol) goat serum for 1 h and incubated overnight with appropriate primary antibodies at 4 °C. Slides were then incubated with Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes for 1 h at 37 °C. Ligation and amplification were carried out using the Duolink detection reagent kit according to manufacturer’s protocol. Images were captured using a confocal microscope. (B) Glioblastoma (U87) and neuroblastoma (SH-SY-5Y) cells were treated with GSK3β inhibitor lithium chloride, and lysates were collected at the indicated periods. Lysates were subjected to Western blot analysis with ^p745PELP1 and total PELP1 antibodies. (C) Coronal brain sections collected from sham, placebo, and E2-treated FLOX mice 6 d after GCI reperfusion were subjected to immunofluorescence for PELP1 antibody.