Phenyl methyl sulfonyl fluoride (pmsf)

The medium was removed and the cells were washed with phosphate-buffered saline (PBS). Cells were collected and lysed using a radioimmunoprecipitation assay lysis buffer (YEASEN, China) with 1/100 PMSF (YEASEN). The total protein of all samples was quantified using a bicinchoninic acid protein assay kit (YEASEN). Each sample containing an equal amount of total protein was separated by 10% SDS-PAGE. All subsequent operations conformed to the standard process. All primary antibodies in this paper were purchased from Abcam (England, United Kingdom). Antibodies used were as follows: anti-β-catenin (Santa Cruz, Dallas, TX, United States, sc7199, 1:1,000), anti-p (S37)-β-catenin (Abcam, ab47335, 1:1,000), anti-GSK3β (Abcam, ab131356, 1:1,000), anti-p (Y216)-GSK3β (CST, 9323s, 1:1,000), anti-vimentin (Proteintech, Chicago, IL, United States, 10366, 1:1,000), anti-E-cadherin (CST, Danvers, MA, United States, 3195, 1:1,000), anti-N-cadherin (CST, 13116, 1:1,000), anti-Snail (Abcam, ab180714, 1:1,000), anti-proliferating cell nuclear antigen (PCNA) (Santa Cruz, SC-25280, 1:1,000), anti-c-MYC (Abcam, ab32072, 1:1,000), anti-ERK1/2 (CST, CST-9102, 1:1,000), and anti-BCL2 (CST, CST-3498, 1:1,000).

The whole hippocampus was dissected and mechanically homogenized, then lysed in appropriate volume of radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) containing phenylmethanesulfonyl fluoride (PMSF) (YEASEN) for 1 h on ice, and cleared by centrifugation for 10 min at 15000 g at 4 °C. Protein concentrations of the lysate were determined using Bradford reagent (Bio-Rad, Hercules, CA, USA).
For co-immunoprecipitation, the lysate was precleared with agarose slurry, and incubated with PRG-1 antibody (1:100, synaptic system), then pulled down by Protein G agarose resin (absin). Finally, beads were suspended with appropriate amount of lysis buffer and analyzed by western blot.
For western blot, tissue lysates or immunoprecipitated samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane (Biosharp). Then membranes were incubated with first antibodies, including PRG-1 (1:3000, synaptic system), P2X₇R (1:1000, abcam), PPP2R2A (1:1000, Cell Signaling), ß-actin (1:5000, MP Biomedicals), and horseradish peroxidase (HRPO)-conjugated secondary antibodies (1:5000, dianova). Finally, membranes were developed by ECL (EpiZyme scientific).

The coimmunoprecipitation assay was performed in HEK 293T cells systems as a previous study (Cheng et al., 2015b (link)). In brief, HEK 293T cells were cultured until 80% confluence and then transfected with duPIAS2-HA and DK212-NP-Flag using Hieff Trans. At 36 h post-transfection, the cells were washed with cold PBS and lysed with an IP Western lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA (Beyotime, China), and 1% PMSF (Yeasen, China) for 20 min on ice. The cell lysate was centrifuged at 14,000 g at 4 °C for 15 min. The supernatant was incubated with 50 μL agarose beads (Thermo Fisher Scientific, United States) and rocked for 6 h at 4°C. The beads were washed with TBST three times after incubation and boiled in 2^∗SDS Buffer (Dingguo, China) for 10 min. Protein samples were subjected to Western blot. NP-Flag and duPIAS2-HA were detected using anti-NP mouse monoclonal antibodies (Sino Biological, China) and anti-HA rabbit polyclonal antibodies (Sigma, United States), respectively. The secondary antibodies were DyLight 800 goat antimouse and antirabbit IgG (H+L) (Thermo Fisher Scientific, United States). The membranes were imaged using an Odyssey infrared imaging system (Li-CoR, United States).

The procedure of protein aggregates was prepared according to a previously reported protocol [65 (link)]. 50 OD₆₀₀ units of 12 h-inductional strains in BMMY were harvested by centrifugation (3000g) for 3 min at 4 °C, and pellets were flash frozen in liquid nitrogen. Pellets were washed with 20 mM potassium phosphate (pH 6.8) and resuspended in buffer II [20 mM potassium phosphate pH 6.8, 1 mM EDTA, 10 mM DTT, 0.1% Tween 20, protease inhibitor cocktail (MedChemExpress), 1 mM PMSF (Yeasen, shanghai, China), 150 U ml⁻¹ lyticase and 1.25 U ml⁻¹ Benzonase (Yeasen, shanghai, China)], incubated for 15 min at 30 °C and chilled on ice for 5 min. After sonication, the samples were collected by centrifugation (200g) for 20 min. The supernatants were diluted to identical protein concentrations and taken as input control (total). Aggregates were sedimented by centrifugation (16,000g) for 20 min at 4 °C. The aggregated proteins were washed twice with buffer II (20 mM potassium phosphate pH 6.8, 2% (v/v) NP-40, 1 mM PMSF, protease inhibitor cocktail), sonicated and centrifuged at 16,000g at 4 °C for 20 min. Protein aggregates were sonicated with rehydration buffer (7 M urea, 2% CHAPS, 2 M thiourea, 20 mM DTT, 1% SDS) [36 (link)], boiled in SDS sample buffer, together with totals separated by 12% SDS-PAGE, and analyzed by Coomassie blue staining and western blotting.

Adherent cells were digested with RIPA buffer (Biotime, Shanghai, China) containing PMSF (Yeasen, Shanghai, China) after two washes with ice-cold PBS. Twenty micrograms of total protein from each sample were separated on SDS–PAGE gels and transferred onto NC membranes (Millipore, Bedford, MA, USA). Then, we used 8% nonfat milk to block nonspecific sites for 1 hour at room temperature. The membranes were then incubated with primary antibodies against vascular endothelial growth factor (VEGF; Servicebio, Wuhan, China), TGFBR1 (Abcam, Cambridge, UK), ID4 (Santa Cruz, Dallas, Texas, USA), C-C motif chemokine ligand 2 (CCL2; Huabio, Hangzhou, China), Smad2 (Santa Cruz, Dallas, Texas, USA), pSmad2 (CST, Danvers, Massachusetts, USA), Smad3 (CST, Danvers, Massachusetts, USA), pSmad3 (CST, Danvers, Massachusetts, USA) and GAPDH (CST, Danvers, Massachusetts, USA) at 4°C overnight. The membranes were washed with TBST 3 times for 10 minutes each and then incubated with a horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibody (CST, Danvers, Massachusetts, USA) at room temperature for 2 hours according to the species of primary antibody. Finally, the ECL Detection Kit (NCM Biotech, Suzhou, China) was used for detection and photography. Images of protein bands were analyzed using ImageJ software.