Anti p smad2 ser465 467

THP-1 cells were suspended in a 100 μL cell lysis buffer containing a phosphatase and protease inhibitor cocktail (Sigma). Protein concentration was measured by BCA protein assay (Thermo) and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An equivalent amount of sample (20 µg protein) was loaded in each well. The protein lysate was then subjected to SDS-PAGE and transferred to Immobilon-P PVDF membrane (PVDF). Blots were incubated overnight in primary antibodies at 4°C. The antibodies used were anti-FAM198B (Proteintech), anti-STAT6, anti-STAT3, anti-STAT2, anti-STAT1, anti-Smad2, anti-P-smad2 (Ser465/467), and anti-P-smad2 (Ser245/250/255), purchased from Cell Signaling Technology (CST). On the following day, blots were incubated with the appropriate HRP-coupled secondary antibodies at 4°C for 2 hours. Chemiluminescence was performed using the ECL detection kit (Bioworlde). Band density was estimated, and protein levels were normalized to GAPDH.

Western blotting was performed to analyze the protein expression as previously described [6 (link)]. Briefly, equal amounts of the protein extracts were loaded and separated by SDS-PAGE and transferred to PVDF membranes. Then, the PVDF membranes were blocked in 5% milk at room temperature for 1 h and incubated with the following primary antibodies (dilution 1:1000) overnight at 4 °C: anti-SM22α (#ab14106), anti-Ang1 (#ab183701), anti-VEGF-A (#ab46154), and anti-Tie2 (#ab24859), which were purchased from Abcam company (Abcam, Cambridge, UK); anti-α-SMA (#19245S), anti-p-VEGFR2 (Tyr1175) (#2478S), anti-VEGFR2 (#2479S), anti-p-Smad2 (Ser465/467) (#3108T), anti-p-Smad3 (#Ser423/425) (#9520T), anti-Smad2/3 (#8685T), VE-Cadherin (#2500S), and anti-GAPDH (#2118S) which were purchased from Cell Signaling Technology (CST, MA, USA); and anti-p-Tie2 (Tyr992) (#AF2424) which was purchased from Affinity Biosciences (Cincinnati, OH, USA). After washing three times, the membranes were incubated with a secondary antibody and then visualized by using Pierce ECL western blotting substrate (Thermo Fisher Scientific). Quantification was performed with ImageJ software (Bethesda, MD, USA)

Total protein was extracted from HR8348 and SW837 cells. Western blotting was performed to detect the expression of target proteins. The primary antibodies including anti‐β‐catenin, anti‐phospho (p)‐β‐catenin (Ser552), anti‐glycogen synthase kinase‐3 beta (Gsk‐3β), anti‐p‐Gsk‐3β (Ser9), anti‐Wnt3a, anti‐Smad2, anti‐p‐Smad2 (Ser465/467), anti‐Smad3, anti‐p‐Smad3 (Ser423/425), anti‐transforming growth factor‐beta (TGF‐β), anti‐LC3A/B, anti‐Beclin‐1, and anti‐P62 antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Anti‐TIPE2 antibody was purchased from Abcam (Cambridge, UK). Anti‐cleaved caspase‐3, anti‐cleaved poly adenosine diphosphate‐ribose polymerase (PARP), and anti‐GAPDH antibodies were purchased from ProteinTech (Chicago, IL, USA). The horseradish peroxidase‐conjugated secondary antibody was purchased from CST. The reaction was visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Rockford, IL, USA). The bands were semi‐quantified using Image J software. The results were normalized to the level of GAPDH.