T pertm tissue protein extraction reagent

Tumor tissue samples were homogenized by using T-PER^TM Tissue protein extraction reagent (cat no.78510) (Thermo Fisher scientific, Grand Island, NY, USA) containing protease inhibitor (cat no. P8340) and phosphatase inhibitor (cat no. P2850) (1:100) (Sigma Aldrich’s. Louis, MO, USA) by ice incubation for 40 min. The supernatant was collected after centrifugation at 14,000 rpm for 20 min. Protein concentration was determined by Bradford assay. Approximately 40–60 µg of protein was loaded on 4–12% SDS PAGE. The proteins were transferred to the nitrocellulose membrane, followed by blocking with 5% BSA in TBST for 2 h. Afterward, the membrane was kept on a shaker overnight with primary antibodies to PDL1, PD1, SOD2, PARP, SET, TGF- β, NF-kB, BCL2, and β-actin. The membranes were washed three times with TBST and then were incubated with horseradish peroxidase-conjugated secondary anti-rabbit and anti-mouse (1:3000) antibodies for 2 h at room temperature, and blots were developed by ECL (Bio-Rad, cat no-1705060) reagent on ChemiDoc^TM XRS+ Imaging system (Bio-Rad). Using densitometry and analysis software (Image J 1.36; Wayne Rasband, National Institutes of Health, MD, USA), the relative band densities were determined [48 (link)].

Frozen colonic tissue was homogenized in T-PER^TM tissue protein extraction reagent with protease inhibitor cocktail from Thermo at pH 7.6 and extracted for 15 min on ice to obtain tissue protein solutions. Then the extracted solutions were centrifuged, and the supernatant was diluted to the same final concentrations of protein based on the BCA assay (Thermo, USA) analysis. The concentration levels of IL-1β, IL-6, and TNF-α were measured by the corresponding ELISA quantitative kits according to the manufacturer's protocols.

Protein extraction was done using a T-PERTM tissue protein extraction reagent (Thermo Scientific, USA) and protein levels determined using a BCA kit (Beyotime, Shanghai, China). Overnight incubation of the membranes at 4°C was done with primary antibodies against TH (1:2000), Bax (1:1000), Bcl-2(1:1000), cleaved Caspase 3 (1:1000), PSD95 (1:1000), p53 (1:1000), SYN (1:1000), JNK (1:500), c-Jun (1:1000), p-JNK (1:500), p-c-Jun (1:1000), as well as p-p53(1:1000) after which they were incubated with secondary antibodies. A LI-COR Odyssey scanner was used to detect fluorescence signal (Biosciences, USA) and image J used to measure the strip optical density.

We referred to the protocol described on the Thermo Fisher website¹ or Affinity Biosciences website². Global brain tissue was dissected from normal aging mice and 3 × Tg-AD mice, and proteins were extracted with radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Scientific^TM T-PERTM Tissue Protein Extraction Reagent, 78510). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes followed by incubation with a horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG secondary antibody (1:10000). The antibodies can be seen in the key resources table. Band intensity was quantified using ImageJ software.

Mouse ears were excised and homogenized with T-PER^TM Tissue Protein Extraction Reagent (ThermoFisher Scientific), supplemented with ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Sigma). Ear homogenates were centrifuged at 15,000 rpm for 30 min at 4 °C and supernatant was collected to measure MIP-2 concentration by sandwich ELISA, using a Quantikine mouse MIP-2 set (R&D Systems, Minneapolis, MN, USA).