Ecl western blotting substrate

The PDLSCs washed with prechilled phosphate-buffered saline (PBS) were lysed in a radioimmunoprecipitation assay buffer (Solarbio Science, China) containing 1% phenylmethylsulfonyl fluoride (Solarbio Science, China) for 5 min at 4 °C, followed by three cycles of 5 s sonication on ice. The cell lysate was centrifuged at 12,000 rpm at 4 °C for 15 min to obtain the protein lysate. The proteins were denatured, separated in a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel, and transferred to a 0.2-μm polyvinylidene fluoride membrane (Millipore, USA). The membrane was blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) at pH 7.2, then incubated overnight at 4 °C with primary antibodies of choice. After washing three times with TBS-T, the membrane was incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies of choice at room temperature and washed again with TBS-T. Protein bands were visualized by an Amersham Imager 600 (General Electric, USA) with ECL Western Blotting Substrate (Biosharp, China). Densitometric quantification of the protein bands was conducted by using ImageJ (National Institutes of Health, USA). All primary and secondary antibodies used for western blot in this study are listed in Table 1.

Cells and tissues were lysed in Radio Immunoprecipitation Assay (RIPA) Lysis Buffer (Beyotime, China) containing protease inhibitors. Then the protein concentration of each sample was measured using a BCA Protein Assay Kit (Beyotime, China). For IBT, 15 µg proteins were separated via SDS-PAGE (12% gel) at 90-120 mV for 90 min and transferred to a polyvinylidene difluoride (PVDF) membrane (Invitrogen, USA) at 300 mA for 60 min. Afterwards, the PVDF membranes were blocked with 2.5% bovine serum albumin (BSA) for 2 h at room temperature and then incubated with specific primary antibodies overnight at 4°C. The primary antibody used in this paper: anti-SLC22A12(14937-1-AP), 1:1,000, Proteintech, China; anti-GAPDH(AC002), 1:5,000, Abclonal, China; anti-PI3K(PAB43806), 1:2,000, Bioswamp, China; anti-p-PI3K(PAB43641-P), 1:2,000, Bioswamp, China; anti-AKT1(A17909), 1:3,000, Proteintech, China; anti-p-AKT1(ab81283), 1:3,000, Abcam, US. Following incubation with the primary antibodies, the membranes were incubated with specie-matched secondary antibodies (AS014/AS003, 1:3,000; Abclonal, China) for 2 h at room temperature following washing with PBST for 30 min. Finally, the protein bands were visualized with Electrochemiluminescence (ECL) Western Blotting Substrate (Ultra sensitivity; Biosharp, China) using ChemiDoc-XRS+ (Bio-Rad, China).

Dorsal CA3 tissues (1 mg) from P12 control and shRNA-virus treated mice were homogenized in RIPA lysis buffer (Yeasen, Shanghai, China) containing cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche Applied Science, Basel, Switzerland) and disrupted by ultra-sonication. Protein samples were separated on 10% tris-glycine gels before being transferred onto PVDF (0.45 μm, Merk Millipore, Darmstadt, Germany) and the PVDF was then blocked in 5% nonfat milk. Immunoblotting was conducted with HRP-conjugated secondary antibodies and ECL Western Blotting Substrate (BioSharp, Beijing). The following primary antibodies were used in this study: rabbit anti-Oxtr (Abclonal, Wuhan, China) and rabbit anti-β-tubulin (Yeasen, Shanghai, China). Loading controls were run on the same gel. Signals were acquired using Amersham Imager 600 (GE Life Science, Boston, USA), and images were analyzed using Fiji ImageJ software.