Ab275876

Six dishes of Mia PaCa-2 cells were prepared. Renewed the medium with 20 μmol/L compound 36l and 1% DMSO to every 3 dishes respectively and incubated for another 2 h. Harvested and combined the cells, then added 500 μL of IP lysate (Beyotime, containing 1% protease inhibitor) and lysed the cells on ice for 30 min. The prepared Protein A/G beads (Santa Cruz) were added into the cell lysates. Shacked the tubes at 4 °C for 1 h followed by a centrifuge at 4 °C, 12,000 rpm, 15 min. Quantify the supernatant by the BCA method and adjust the total protein concentration to 5 μg/mL. Respectively added KRAS primary antibody (Abcam #ab275876) and IgG (Abcam #ab125938) antibody to 100 μL the above lysates followed by rotating at 4 °C for 1 h. Add the Protein A/G beads 20 μL respectively into the supernatant above, and rotated on the shaker overnight. Centrifuged and collected the precipitate followed by washed it with 500 μL PBS buffer 3 times. Centrifuged and collected the precipitate followed by denaturation in 2 × loading buffer 60 μL at 95 °C for 10 min. Western blotting experiments and gray-scale statistics were conducted to quantify the KRAS and PDEδ protein.

The expression of related proteins in lentivirus-transfected AGS and HGC-27 cells was detected. Firstly, total protein was extracted with RIPA (Epizyme Biotech, China) from GC cells and their content was confirmed using the BCA assay (Epizyme Biotech, China). Next, the proteins were isolated and transferred onto a polyvinylidene fluoride membrane, which was sealed with 5% skim milk powder, soaked in primary antibody and incubated overnight. On the second day, it was washed with TBST and soaked in HRP-linked secondary antibody (1:1000, 7074/7076, CST, United States) for 1 h. Finally, protein bands were visualized with ECL reagent (KeyGEN BioTECH, China), and the gray values of the protein bands were analyzed using Image J. GAPDH or β-tubulin was used as an internal control to standardize target proteins. p-ERK1/2 (1:5000, 4370), ERK1/2 (1:5000, 9102), RIP1 (1:1000, 3493), p-RIP1 (1:1000, 65746), MLK1 (1:1000, 5029) and p-MLK1 (1:1000, 91689) antibodies were purchased from CST. BAX (1:2000, ab32503), Caspase-3 (1:1000, ab13847), BCL-2 (1:5000, ab182858) and KRAS (1:1000, ab275876) antibodies were purchased from Abcam.