Rabbit anti kras

Cells were sonicated or homogenized in RIPA buffer (20 mM Tris‐HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP‐40, 0.1% SDS, and 1% sodium deoxycholate). After being clarified by centrifugation at 12 000 g for 15 min at 4 °C, the supernatant was collected for Bradford protein assay. Western blotting was performed as previously reported.^[58 (link)
^] Briefly, equal molarity of protein extracts was loaded and separated in an SDS–PAGE, and transferred to a PVDF membrane. After being blocked with 5% skimmed milk at RT for 1 h, the membrane was incubated with primary antibodies (rabbit anti‐MUC4 (1:1000; Thermo Fisher Scientific #35‐4900), ribbit anti‐GFP (1:10 000; GeneTex #GTX113617), rabbit anti‐KRAS G12D (1:1000; Cell Signaling Technology #14 429), rabbit anti‐KRAS (1:2000; Cell Signaling Technology #67 648), rabbit anti‐Activin A (1:1000; GeneTex #GTX108405), mouse anti‐GAPDH (1:10 000; GeneTex #GTX627408) at 4 ˚C overnight and treated with Goat Anti‐Rabbit IgG (HRP) (1:3000; GeneTex #GTX213110‐01) and Goat Anti‐Mouse IgG (HRP) (1:3000; GeneTex #GTX213111‐01) antibodies at room temperature for 1 h. Chemiluminescent detection of the horseradish peroxidase reaction was performed using Immobilon Forte Western HRP substrate (Merck #WBLUF0500) according to the manufacturer's instruction and filmed by ChemiDoc MP Imaging System (Biorad).

Cells were washed three times in cold PBS, and protein lysates were obtained by extraction with RIPA buffer containing protease inhibitors (Sigma). Proteins were separated on 10% pre-cast SDS-PAGE gels (Bolt™ 4–12% Bis-Tris Plus Gels) (Invitrogen), and were then transferred using the iBlot system (Life Technology). Membranes were incubated with the following primary antibodies: rabbit anti-KRAS, anti-phospho-p42/44 MAPK (ERK1/2), anti-p42/44 (ERK1/2), anti-phospho-Akt, anti-phospho-p38 MAPK, and anti-p38 (all from Cell Signaling, Danvers, MA; used at 1:1000 dilution); rabbit anti-Akt (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA); and mouse anti-β-actin (1:4000, Sigma). Membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies (Pierce Biotechnology Inc., Rockford, IL, USA), and detected using a SuperSignal^® West Femto enhancer kit (Pierce). Protein intensity was analyzed by Image J (NIH, Bethesda, MD, USA) in triplicate. The ratios of KRAS/β-actin, phosphor-AKT/AKT, phosphor-p42/44/p42/44 andphosphor-p38/p38 were calculated. The expression level was compared to no treatment group.