Rack1

Antibodies against the following proteins were used: RACK1 (1:1000, Cell Signaling, Danvers, MA, USA), phospho-Akt (Ser473) (p-Akt, 1:1000, Cell Signaling), Akt (1:1000, Cell Signaling), phospho-FAK (Tyr397) (p-FAK, 1:1000, Cell Signaling), FAK (1:1000, Cell Signaling), HA (1:1000, Cell Signaling), PHB2 (1:500, Santa Cruz, CA, USA), integrin β1 (1:500, Santa Cruz), β-actin (1:1000, Santa Cruz) and β-tubulin (1:500, Proteintech, Wuhan, China). Horseradish peroxidase-conjugated secondary antibodies (anti-mouse/rabbit IgG) (1:5000, Cell Signaling) were used.

Cells were cultured in six‐well plates with SBSGL at 0, 600, 1200 and 1800 μg/mL concentrations. Radio immunoprecipitation assay buffer (Shanghai Biyuntian Biotechnology Co., lot no. P0013C) with a protease inhibitor was used to extract proteins. Equal amounts of protein from each sample were transferred to polyvinylidene fluoride membranes (Merck, lot no. IPFL00010). Membranes were incubated with primary antibodies against RACK1 (Cell Signaling Technology, lot no. 5432, 1:1000), OGT (Cell Signaling Technology, lot no. 24083, 1:1000), O‐GlcNAc (Thermo Fisher Scientific, lot no. MA1‐072, 1:1000), LC3B (Cell Signaling Technology, lot no. 3868, 1:1000), p62 (Cell Signaling Technology, lot no. 5114, 1:1000), PD‐L1 (Cell Signaling Technology, lot no. 13684, 1:1000), Myc‐tag (Cell Signaling Technology, lot no. 2276, 1:1000) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, Cell Signaling Technology, lot no. 5174, 1:1000) overnight at 4°C. After washing with Tris‐buffered saline with Tween (TBST, Biosharp, lot no. 21259419), secondary antibodies goat anti‐rabbit IgG‐HRP (Abbkine, lot no. A21020, 1:10,000) and goat anti‐mouse IgG‐HRP (Abbkine, lot no. A21010, 1:10,000) were applied for 1 h at room temperature. Membranes were washed thrice with TBST, and visualization was achieved using an ECL luminescence kit (Shanghai Biyuntian Biotechnology Co., lot no. P0018S).

Protein lysates were prepared from olfactory bulbs and hippocampi of 5 months old WT and KO female mice using RIPA buffer with protease inhibitors followed by sonication by ultrasound and clearing via centrifugation at maximum speed using Eppendorf tabletop centrifuge. Protein concentration was quantified using BCA reagent (Thermo Fisher Scientific, Rockford, IL). Protein extracts (40 μg/well) were subjected to Western immunoblot analysis. We used antibodies against the following proteins: PRDX1 (Sigma), ACC1, pACC1, AMPKα, pAMPKα, NGFR, TrkB, Bcl-xL, Rack1 (all from Cell Signaling Technology), Calretinin (Millipore), LC3 (Abcam). The band intensity was determined using ImageJ software, normalized to loading control (β-actin). The data are presented as a relative intensity of WT bands over KO bands. Phospho-index was determined as a ratio of phosphorylated/total protein intensities.