Mouse anti gnao1

Cells or minced tissues were lysed in RIPA buffer (Beyotime, Shanghai, China) and centrifuged at 13,200 rpm for 15 min at 4 °C. After the quantification of protein concentration (Beyotime), WB was performed by following the same procedure described previously, except for the primary antibodies: Rabbit anti-MAG (1:100; Invitrogen, 34-6200), Mouse anti-Gnao1 (1:1000; Santa Cruz, sc-13532), Rabbit anti-Gnao1 (1:1000; Abcam, ab154001), Rabbit anti-AKT (1:1000; CST, 9271S), Rabbit anti-pAKT (1:1000; CST, 9272S), Rabbit anti-ERK1/2 (1:1000; CST, 9102S), Rabbit anti-pERK1/2 (1:1000; CST, 9101S), Rabbit anti-PI3K (1:1000; CST, 4292), Rabbit anti-pPI3K (1:1000; CST, 17366), and Mouse anti-GAPDH (1:50,000; Proteintech, Chicago, USA, 60004-1-Ig).

Mice were perfused intracardially with 4% paraformaldehyde (PFA) and the PFA-post-fixed tissues were cryoprotected in optimal cutting temperature compound (OCT) and processed for 12 µm cryosections for IHC. ICC is used for PFA-fixed cells. Sections or PFA-fixed cells were permeated in 0.2% Triton-X100 in phosphate buffered saline (PBS), blocked with 5% donkey serum for 1–2 h at room temperature (RT), and incubated overnight at 4 °C with primary antibodies. Sections or cells were then washed thrice before corresponding fluorescence-conjugated secondary antibody (1:1000; Jackson Immunoresearch, PA, USA) incubation for 1 h at RT, following by three washes and mounting with coverslips. Images were captured using a fluorescence microscope (Leica). Primary antibodies used are: Mouse anti-S100β (1:200; Sigma, S2532), Rabbit anti-MAG (1:100; Invitrogen, 34–6200), Mouse anti-Gnao1 (1:1000; Santa Cruz, CA, USA, sc-13532), Rat anti-F4/80 (1:200; Abcam, ab6640), Mouse anti-β-tubulin-III (1:1000; Sigma, T8578).