Ripa protein extraction kit

Protein extraction from tissue samples was performed using the RIPA Protein Extraction Kit (Thermo Fisher Scientific, USA) following the operation manual. Western blotting was performed as described in the literature [11 (link)]. The extracted proteins were subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. After blocking with 5% milk for 1 h, the membrane was incubated with the primary antibody at 4°C overnight. The primary antibodies included anti-NF-κB (catalog no. Ab16502, Abcam, Cambridge, UK, 1 : 1000 dilution), anti-NF-κB (phospho S536) (catalog number ab86299, Abcam, Cambridge, 1 : 1000 dilution), and anti-b-GAPDH (sc-47778, Santa Cruz Biotechnology, 1 : 1000 dilution). After incubation, the PVDF membrane was rinsed and then incubated with the secondary antibody for 1 h at room temperature. Then, the protein expression was detected by using a chemiluminescence detection system (Millipore, Billerica, MA, USA).

The colon tissues were pestled into powder in liquid nitrogen, and the total proteins were extracted with ice-cold RIPA protein extraction kit (Thermo Fisher scientific, MA, USA). After centrifugation at 10,000 × g at 4°C for 10 min, the supernatants were collected to obtain the protein extracts and the concentration of each sample was determined with BCA assay kit (Jiancheng Bioengineering Institute, Jiangsu, China). The proteins were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to the polyvinylidene fluoride (PVDF) membrane (0.45 mm, Millipore, MA, USA). The proteins were incubated at 4°C overnight with STAT3 (STAT3, JAK1, JAK2, and JAK3), MAPK (p-ERK, ERK, p-JNK, JNK, p38 MAPK, and p-p38 MAPK), or NF-κB (NF-κB p65 and IκB-α) primary antibodies (1 : 1000, Cell Signaling Technology, KY, USA) and then incubated with corresponding secondary antibody (1 : 4000) at room temperature for 1 h. The membranes were washed for 3 times with Tris-buffer [contain 0.1% (v/v) Tween^® 20] and incubated with enhanced chemiluminescence (ECL) reagent (Millipore, MA, USA). The protein bands were captured with X-ray films and quantified by Imaging System (Bio-Rad, Hercules, CA) using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal reference control.