Ab32391

Synthetic human Aβ_1‐42 peptide was purchased from GL Biochem , Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco , mouse recombinant Wnt2b (rWnt2b) was purchased from Cusabio, 1,1,1,3,3,3‐Hexafluoro‐2‐propanol (HFIP) was purchased from Sigma‐Aldrich . The primary antibodies used were rabbit anti‐Wnt2b (ab178418; Abcam), mouse anti‐β‐catenin (ab32572; Abcam); rabbit anti‐phospho‐β‐catenin (Thr41/Ser45) (9565 S; Cell Signaling Technology); rabbit anti‐GSK3β (ab32391; Abcam); rabbit anti‐phospho‐GSK3β (Ser9) (ab75814; Abcam); rabbit anti‐BDNF (ab108319; Abcam); rabbit anti‐SDHB (A10821; ABclonal); mouse anti‐β‐actin (ab6276; Abcam); The secondary antibodies used were Goat Anti‐Rabbit IgG (HRP) (ab205718; Abcam); Goat Anti‐Mouse IgG (HRP) (ab6789; Abcam); Donkey anti‐Rabbit IgG (ab150073; Abcam).

Immunohistochemistry was performed as previously described [16 (link)]. Briefly, sections containing auditory cortex were double-immunostained with rabbit anti-GSK3α (1:200, G08-63R-25, SignalChem, USA), rabbit anti-GSK3β (1:1000, ab32391, Abcam, USA), or rabbit GSK3β phospho-Y216 (1:150, ab75745 Abcam, USA), and mouse anti-PV (1:5000, 235, Swant, Switzerland). Images of the auditory cortex were captured using confocal microscope (Nikon A1). NIH ImageJ software was used to measure the integrated density of GSK3α, GSK3β and GSK3-phospho (Y279/Y216) in PV-positive interneurons or non-PV neurons. The corrected total cell fluorescence (CTCF) was calculated as previously described [26 (link)]. Results are presented as normalized florescence, which is the ratio of CTCF value of the protein to the CTCF values of corresponding proteins in non-PV neurons of floxed-control mice.

Total protein in tissues or cells was extracted with radio immunoprecipitation assay (RIPA) lysis buffer containing phenylmethanesulfonyl fluoride (P0013C; Beyotime Institute of Biotechnology), with the concentration detected by BCA kit. After sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), the protein was electrotransferred onto a polyvinylidene fluoride membrane and blocked with 5% skim milk powder at room temperature for 1 hour. Then, the membrane was probed with diluted primary antibodies to GSK3β (1:5000, ab32391; Abcam), FTO (1:1500, ab126605; Abcam), MZF1 (1:500, ab64866; Abcam,), c‐Myc (1:1000, ab32072; Abcam), CDK2 (1:1000, ab32147; Abcam), CDK4 (1:500, ab137675; Abcam), Ki‐67 (1:1000, ab16667; Abcam), PCNA (1:1000, ab18197; Abcam), Bax (1:1000, ab199677; Abcam), Bcl‐2 (1:500, ab59348; Abcam) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (ab9485, 1:2500; Abcam) at 4°C overnight. Next day, the membrane was re‐probed with horseradish peroxidase (HRP)‐labelled secondary antibody of goat anti‐rabbit antibody to immunoglobulin G (IgG) for 1 hour and visualized by enhanced chemiluminescence (ECL) kit (BB‐3501, Ameshame; Chiltem). Finally, Quantity One v4.6.2 software was used to quantify the grey levels of each band in the Western blot image. GAPDH served as an internal reference.

Protein solutions were extracted from hippocampal tissues; then, equal amounts of protein were attached onto a sodium dodecyl sulfate-polyacrylamide gel to be separated by electrophoresis according to their molecular weight. After electrophoresis, proteins were transferred to a nitrocellulose membrane (Amersham Bioscience, Piscataway, NJ, USA) using semidry transfer apparatus (Bio-Rad, Hercules, CA, USA). The membranes' nonspecific binding sites were then blocked by soaking in 5% skimmed milk. Next, the membranes were incubated at 4°C overnight on a roller shaker with solutions containing antiphosphorylated tau (1 : 10000, Cat. # ab109390), anti-GSK-3β (1 : 5000, Cat. # ab32391), and anti-mTOR (1 : 10000, Cat. # ab134903), which were obtained from Abcam (Cambridge, MA, USA). The membranes were then washed and incubated with the horseradish peroxidase-conjugated secondary antibody solution. Finally, the blots were developed with enhanced chemiluminescence detection reagents (Amersham Biosciences, Arlington Heights, IL, USA). Scanning laser densitometry (GS-800 system, Bio-Rad, Hercules, CA, USA) was used to determine the quantities of the target proteins. The results were normalized with β-actin protein expression and expressed as arbitrary units.

Tissues were lysed firstly, and protein content was detected using bicinchoninic acid kit (BCA, #P0012S, Beyotime, China) assay. Same amount of protein was separated by 10% SDS-PAGE and transferred to nitrocellulose membrane (Invitrogen, USA). The membrane was blocked with TBST solution (5% skim milk) for 2 h, and cultured with primary antibodies (1:800) at 4°C overnight. After washing twice using PBS, a secondary antibody (1:2000) was used to incubate proteins for 1 h. ImageJ software was applied to calculate the protein bands grey. The antibodies details were listed as follows: anti-beta Catenin antibody (ab32572, ABCAm, Cambridge, UK), anti-GSK3 beta antibody (ab32391, ABCAm, Cambridge, UK), and goat anti-Rabbit IgG (ab205718, ABCAm, Cambridge, UK).

Total protein was extracted from LUAD cells using RIPA buffer (Beyotime, Beijing, China) containing protease inhibitor and phosphatase inhibitor. The protein concentrations were detected by BCA assay (Beyotime, Beijing, China). An equal amount of protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, CA, USA). After blocking with 5% skimmed milk for 2 h, the membranes were incubated with primary antibodies specific for UBE2T (1:1000, ab140611, Abcam), FBLN5 (1:500, ab66339, Abcam), Bcl-2 (1:2000, ab182858, Abcam), Bax (1:1000, ab32503, Abcam), cleaved-caspase-3 (1:500, ab32042, Abcam), pro-caspase-3 (1:1000, ab32499, Abcam), p-ERK (1:2000, Cell Signaling Technology, #4370), ERK (1:1000, Cell Signaling Technology, #4695), p-GSK3β (1:2000, ab75814, Abcam), GSK3β (1:2000, ab32391, Abcam), β-catenin (1:1000, Cell Signaling Technology, #8480), β-actin (1:1000, ab8227, Abcam) at 4°C overnight, followed by HRP-conjugated secondary antibody (1:2000, ab205718, ab6789, Abcam) for 1 h. The positive bands were detected by using an Enhanced Chemiluminescence Kit (Thermo Fisher Scientific) [32 (link)].