Anti phospho mek1 2

LUAD cells were lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors. Cell lysates were electrophoresed in 12% SDS-PAGE and then transferred onto a nitrocellulose membrane (MerrckMillipore, Ireland). Membranes were blocked with TBST (Tris-buffered saline, 0.1% Tween-20) containing 5% skim milk for 2 h and were incubated with the indicated antibodies, including anti-MEK1/2 (Abcam, CAT# ab178876);anti-Phospho-MEK1/2 (CST, CAT# 9154 T);anti-ERK1/2 (CST, CAT#4695 T); anti-Phospho-ERK1/2(CST, CAT#4370 T); anti-AKT (CST, CAT#4691 T); anti-Phospho-AKT (CST, CAT#4060 T);anti-STAT3 (Huaan Biotech, ET1607-38); anti-Phospho-STAT3 (CST, CAT#9145 T) overnight at 4 °C. Then, the membranes were washed with TBST and incubated with a peroxidase-conjugated second antibody for 1 h. The protein bands were detected by enhanced chemiluminescence.

Protein was extracted using SDS lysis buffer (Beyotime Biotechnology, Shanghai, China), and equal amounts of total protein were separated by 10% SDS–PAGE and then transferred to nitrocellulose membranes (Millipore, USA). The membrane was blocked with 5% nonfat powdered milk (Sangon, China) in TBST at room temperature for one hour, incubated with primary antibodies overnight at 4 °C, and then washed with TBST followed by a 1 h incubation with secondary antibodies. The protein bands were visualized by an enhanced chemiluminescence detection kit (Tanon, China). Semiquantitative analysis of the protein density by western blotting was performed using ImageJ (Version 1.5.3). The following antibodies were used at a 1:1000 dilution: anti-SLC25A5 (Cat. #14671, Cell Signaling), anti-SLC25A24 (Cat. #221120, Abcam), anti-COX IV (Cat. #202554, Abcam), anti-phospho-MEK1/2 (Cat. #178876, Abcam), anti-MEK1/2 (Cat. #278564, Abcam), anti-phospho-ERK1/2 (Cat. #201015, Abcam), anti-ERK1/2 (Cat. #184699, Abcam), anti-caspase-1 (Cat. #207802, Abcam), anti-IL-1β (Cat. #254360, Abcam), anti-IL-18 (Cat. #243091, Abcam), anti-gasdermin D (Cat. #219800, Abcam), anti-β-actin (Cat. #4970, Abcam) and anti-GAPDH (Cat. #2118, Cell Signaling).

The protocol for Western blotting is described in detail in [18 (link)]. The following antibodies were used: anti-ERK1,2 (Santa Cruz Biotechnology, Heidelberg, Germany; sc-94), anti-phospho-ERK1,2 (Cell Signaling Technology (CST), Frankfurt am Main, Germany; no. 9101), anti-MEK1,2 (CST; no. 9122), anti-phospho-MEK1,2 (CST; no. 9154), anti-HA-tag (Abcam, Cambridge, UK; ab9110), anti-tet-Repressor (Merck, Darmstadt, Germany; AB3541), anti-HSP90β (Enzo Life Sciences, Lörrach, Germany; ADI-SPA-843), anti-α-tubulin (Bio-Rad, München, Germany; MCA78G). Secondary antibody F(ab')2 fragments coupled to horseradish peroxidase and specific for rabbit-IgG (no. 111-036-045), mouse-IgG (no. 115-036-072) or rat (no. 112-036-062) were obtained from Jackson ImmunoResearch, Newmarket, UK.
A freshly made solution of luminol (2.5 mM), p-coumaric acid (0.2 mM) and H₂O₂ (0.01%) in 100 mM Tris-HCl (pH 8.8) was used as reagent for chemiluminescent detection [19 (link)].