Bca kit

For cell protein extraction, the lysate was SDS lysate with added protease inhibitors and phosphatase inhibitors (EpiZyme, Shanghai, China). Proteins were quantified using a BCA kit (EpiZyme). In each lane, 20 µg of protein lysate was loaded; for phosphorylated proteins with a lower abundance, 40 µg of protein lysate per lane was introduced. We used SDS-PAGE (EpiZyme) gels to separate the prepared protein samples and subsequently transferred them onto 0.25 μm polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany) using electroblotting. After that, we blocked the membranes in 5% milk for one hour, rinsed them three times with Tris Buffered Saline with Tween (TBST) (Servicebro, Wuhan, China), and exposed them to the corresponding primary antibody overnight. The following day, we washed the membranes three times with TBST and then treated them with the Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, #SA00001-2; #SA00001-1; proteintech) for one hour. The bands were then detected using chemiluminescent detection reagents (Beyotime, Shanghai, China). Information on the primary antibodies is shown in Table 3.

Liver tissue was homogenized in 150 mM ice-cold PBS (pH 7.2) containing 1 mM EDTANa2 to prepare a 10% liver homogenate, and this was centrifuged at 1000× g for 10 min at 4 °C. The supernatant was taken to test the XOD and ADA enzyme activity using commercial kits (Jiancheng Biotech, Nanjing, China). Protein levels were determined by the BCA kit (Epizyme, Shanghai, China) using Bicinchoninic Acid with bovine serum albumin as a standard. Values were normalized to liver total protein.

After various treatments as indicated, cancer cells were harvested, and the total protein was lysed in radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) and then quantified by a BCA kit (Epizyme, Shanghai, China). Equal amounts of proteins were resolved on 10%-12.5% SDS-PAGE gels, followed by transferring the proteins to an Immobilon PVDF Membrane (Merck Millipore Ltd, Tullagreen, Ireland). PVDF membranes with proteins were blocked with 5% skim milk for 1 hour, incubated with primary antibodies overnight at 4°C, and then conjugated with secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 hour. Signals were visualized by ECL reagent (Epizyme, Shanghai, China) and photographed by Tanon 5200 visualizer (Tanon, Shanghai, China).
The primary antibodies used are as follows: p27, p21, Cyclin D1, PARP, cleaved PARP (c-PARP), cleaved caspase 3, cleaved caspase 9, cleaved caspase 7, cleaved caspase 8, Noxa, Bak, Bax, Bik, Bim, Bad, Puma, Bcl2, Bcl-xl, Mcl-1, XIAP, BID, TRAIL, DR4, DR5, CHOP, ATF4, eIF2α, and p-eIF2α were from Cell Signaling Technology (USA). Cyclin E, CDK2, CDK4, CDK6, Fas, DR3, c-Myc, and p53 were from Santa Cruz Biotechnology (USA). TNFR1 and TNFR2 were from Proteintech (USA). β-Actin was from HuaBio (China).

The expression of IL-6 and TNF‐α in rat colon tissues was determined by using ELISA Kit (RayBiotech, USA) according to the manufacturer’s instructions. The rat colon tissues were lysed with 2X Lysis Buffer (RayBiotech, USA) adding a complete protease inhibitor cocktail (Epizyme, CN). The tissue homogenates were centrifuged at 12,000 g at 4 °C for 10 min, and the supernatant containing proteins was collected. Then, protein concentration was determined with a BCA kit (Epizyme, CN). Then add 100 µL samples into the appropriate wells, and finally detected the OD value at 450 nm by the microplate reader. The concentration of target protein was calculated according to the standard curve and total protein concentration.

Skin lesions of 3 healthy individuals and 3 patients with psoriasis were selected (including before and after treatment). Total protein was extracted from the tissue using RIPA buffer (Epizyme, Shanghai, China) and measured using a BCA kit (Epizyme, Shanghai, China). Protein samples were isolated using 10% SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with 5% skim milk for 2 h. The main antibodies (Anti-CDK1, Rabbit, Cambridge, UK; anti-CCNB1, Rabbit, Abcam, Cambridge, UK) were at dilutions of 1:20000 and 1:50000, respectively. Image density analysis was performed using ImageJ software (NIH Image, Bethesda, MD).

RIPA buffer (PC104, Epizyme, China) was applied to extract protein from GBM tissues followed by centrifugation. The lysed protein was quantified utilizing BCA kit (ZJ101, Epizyme, China). After electrophoresis (10% SDS-PAGE), the protein was blotted onto nitrocellulose membranes. After being sealed, the membranes were immersed in primary antibodies at 4°C overnight. A further 60 min reaction was done after the addition of secondary antibodies. After being developed with an ECL luminescence reagent (P1000, Applygen, China), the signals were analyzed by a gel imaging system. The primary antibodies of Cyclin D1 (1 : 200, ab16663), C-myc (1 : 1000, ab32072), Bad (1 : 1000, ab32445), p-p53 (1 : 5000, ab33889), ATM (1 : 1000, ab201022), Histone H2A.X (1 : 1000, ab20669), p-Histone H2A.X (1 : 1000, ab81299), phospho-STAT3 (Ser727) (1 : 1000, ab32143), phospho-STAT3 (Tyr705) (1 : 10000, ab267373), STAT3 (1 : 2000, ab68153), and GAPDH (1 : 10000, ab181602) were gained from Abcam (UK). The primary antibodies of p-AKT (1 : 2000, #4060), AKT (1 : 1000, #4685), Cleaved Caspase-9 (1 : 1000, #9507), and p-ATM (1 : 1000, #2851) were obtained from CST (USA). PD-L1 (1 : 1000, abx179111) was bought from Abbexa (USA). Pro-Caspase-9 (1 : 2000, AF6348) was bought from Affinity (USA).

Liver and intestinal samples were homogenized in RIPA buffer added with protease and phosphatase inhibitors. Full centrifugation at low temperature (15 min at 12,000 g) to obtain supernatant, and the protein concentration was quantified by BCA kit (Epizyme, Shanghai, China), proteins electrophoresis using the 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto 0.45 μm PVDF membranes (Millipore, United States). Subsequently, the PVDF membranes were socked in 5% skim milk containing 140 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.5), and 0.1% Tween 20°at room temperature for 60 min, and incubated with primary antibodies at 4°C overnight: FXR mouse monoclonal antibody (72105S, CST, United States), TGR5 rabbitpolyclonal antibody (72,608, Abcam, United States), ZO-1 (ab216880, Abcam, United States), Occludin (ab 216,327, Abcam, United States), Claudin 2 (ab53032, Abcam, United States), P-P65 rabbit monoclonal antibody (3031S, CST, United States), P65 rabbit monoclonal antibody (8242S, CST, United States), β-actin (Hua-an Biotech Inc., Hangzhou, China), and then incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for another 60 min. The protein bands were visualized by an ECL chemiluminescence detection kit (WBKLS0500, Millipore, United States) with an enhanced chemiluminescence system (Tanon 5200, Shanghai, China).