Ac033

For puromycin incorporation, overnight cultures were diluted to an OD₆₀₀ of 0.1 to 0.2 with 5 mL of fresh medium with or without 1 mg/mL paromomycin (Solarbio, P8230) treatment for 6 h and grown to log phase. puromycin was added directly to the culture at a final concentration of 2 mM and incubated 1 h at 30 °C. Then, two OD₆₀₀ cells were collected and lysed by alkaline lysis. The sediment was suspended in SDS buffer (60 mM Tris- HCl [pH 6.8], 10% glycerol, 2% SDS, 0.01% bromophenol blue, 0.01 M DTT, and 5% β-mercaptoethanol) and heated at 100 °C for 10 min. The protein samples were collected form the supernatant after centrifuging. Equal volumes of protein samples were separated on 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels, followed by detection of puromycin-incorporated proteins with the anti-puromycin monoclonal antibody (Merck, MABE343). Equal loading was controlled by the protein level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ABclonal, AC033).

The tissues were homogenized in ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) and supplemented with complete EDTA-free protease inhibitor cocktail and PhosSTOP Phosphatase Inhibitor. The protein samples were mixed with 5 × dual color protein loading buffer (Fudebio, Hangzhou, China) and boiled at 98 °C for 10 min. Equal amounts of protein (30 μg/lane) were loaded on a SDS-PAGE gel at a constant voltage and then transferred to a polyvinylidene difluoride membrane (Millipore, MA, USA). After being blocked and incubated with the primary and secondary antibodies, the bands were visualized using a chemiluminescence image analysis system (Tanon, Shanghai, China) with FDbioFemto ECL (Fudebio, Hangzhou, China). The band intensities were quantified using Gel-Pro Analyzer software (Media Cybernetics, Maryland, USA). The primary antibodies were included the following: synaptosomal associated proteins 25 (SNAP-25; Abcam, ab109105), postsynaptic density proteins 95 (PSD-95; CST, 3450), Ibα1 (Abcam, ab178846), TNF-α (Abcam, ab215188), IL-6 (Abcam, ab233706), NLRP3 (Abcam, ab263899), IL-18(Abcam, ab243091), IL-1β (Abcam, ab254360), caspase-1 (Abcam, ab207802), TLR4 (Abcam, ab22048), myeloid differentiation factor 88 (MyD88; Abcam, ab133739), NF-κB p65 (Abcam, ab32536), GAPDH (ABclonal, AC033), ZO-1 (ABclonal, A11417), occludin (ABclonal, A12621) and β-actin (ABclonal, AC026).

The reagents used for study included RIPA buffer (high) (R0010, Solarbio, Beijing, China), BCA protein assay kit (P0010, Beyotime Biotechnology, Shanghai, China), phosphatase inhibitor (M7528, AbMole, Shanghai, China), anti-CD34 antibody (GB121693, Servicebio, Wuhan, China), anti-PI3K antibody (4249, Cell Signaling Technology (CST), USA), anti-AKT antibody (4691, CST, USA), anti-pAKT antibody (4060, CST, USA), anti-VEGF antibody (A5708, ABclonal, Wuhan, China), GAPDH antibody (AC033, ABclonal, Wuhan, China), goat anti-rabbit IgG (AS014, ABclonal, Wuhan, China) and goat anti-mouse IgG (AS003, ABclonal, Wuhan, China).
The instruments employed used included a light microscope (AxioScope.A1, Zeiss, Germany), photomicroscope (AxioVert.A1, Zeiss, Germany), slice scanner (KF-PRO-005-EX, Ningbo, China), high-speed low-temperature tissue grinding instrument (KZ-III-F, Servicebio, Wuhan, China) and electrophoresis system (Mini-Protean system, BioRad, USA).