Bca kit

The cells under the described treatments were lysed in RAPI (protein lysis buffer containing protease inhibitor and phosphatase inhibitor) for 30 min on ice. The obtained proteins in the supernatants were then quantified using a BCA kit (Tiangen, Beijing, China). Equal amounts of protein sample (50 µg) were separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis, thus electrically transferring the proteins onto polyvinylidene fluoride membranes (Millipore, Boston, MA). Then, the resultant membranes were blocked with 5% skim milk in TBST buffer for 1 h at room temperature prior to coincubation with the corresponding primary antibodies against TIMP1 and TIMP2 (Proteintech, Wuhan, China) or Notch-1 (Santa Cruz Biotechnology) overnight at 4 °C. Subsequently, the membranes were incubated with secondary antibody (Jackson, West Grove, USA) for 1 h at room temperature. The gray levels of proteins were visualized with horseradish peroxidase-conjugated secondary antibodies via exposure to a chemiluminescence (ECL) detection kit (Solarbio, Beijing, China). The images were obtained using Quality One Imaging software (Bio-Rad, USA). The monoclonal GAPDH antibody (Proteintech, Wuhan, China) served as an internal control.

Western blot analysis was performed as the previously reported method [22 (link)] with slight modifications. The total proteins of colon tissue were extracted with RIPA buffer (the mass ratio of sample:RIPA = 1:10) and supplemented with 1× protease inhibitor cocktail (Roche, Basel, Switzerland) on an ice bath. Then, the supernatant was collected after centrifugation (10,000× g for 10 min at 4 °C). The concentration of the protein was measured using the BCA kit (Tiangen Biotech, Shanghai, China). The protein samples of the same amount (loading amount of 40 µg) were separated on a 12% SDS-PAGE gel and transferred onto a PVDF membrane (Sigma Aldrich, Shanghai, China). After being blocked with TBST (tris-buffered saline containing 0.1% Tween 20) containing 5% skimmed milk, the membrane was incubated overnight at 4 °C with primary antibodies (NF-κB p65, 1:2000; IκB-α, 1:2000; β-actin, dilution 1:100,000), and incubated with secondary antibodies (dilution 1:5000) for 2 h at room temperature after washing. Protein bands were detected using the ECL Plus™ Western Blot Detection System (Pierce, Rockford, IL, USA) and imaged with a GIS V2.0 imaging system (Tannon, Shanghai, China).

The samples were ground to powder with liquid nitrogen and then added to a four-volume cracking buffer followed by ultrasonic treatment on ice. An equal volume of Tris-Balanced phenol was added to remove residual debris by centrifugation. Five volumes of 0.1 M ammonium acetate/methanol were then added to precipitate the protein overnight. The protein was redissolved in 8 M urea, and the protein concentrations were detected with a BCA kit (PA115, Tiangen, China).
The protein solution was reduced with 5 mM dithiothreitol at 56 °C for 30 min, followed by alkylation with 11 mM iodoacetamide under dark conditions for a further 15 min. The protein sample was then diluted to a urea concentration of no more than 2 M by the addition of 100 mM TRAB. Lastly, trypsin was added at a 1:50 mass ratio of trypsin to protein and incubated for 4 h at a mass ratio of 1:100 trypsin to protein.

Cells were harvested and lysed using RIPA buffer (Thermo Fisher Scientific) in the presence of the protease inhibitor cocktail (Sigma‐Aldrich) at 24 hours after LED irradiation. Proteins were then quantified with a BCA kit (PA115; Tiangen Biotech, Beijing, China), resolved on 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels, and transferred onto polyvinylidene difluoride (PVDF) membranes (162‐0177; Bio‐Rad, Hercules, CA, USA). Membranes were incubated with the appropriate primary antibody followed by incubation with the corresponding horseradish peroxidase (HRP)‐conjugated secondary antibody, and proteins were detected with enhanced chemiluminescence (ECL) Western blotting substrates (W1001; Promega, Madison, WI, USA).