Anti mek1 2

Proteins were extracted from lung tissue and cell samples through homogenization in RIPA lysis and extraction buffer (ThermoFisher Scientific) according to the manufacturers’ instructions. After denature in SDS loading buffer, protein samples were separated by 8% or 10% SDS-PAGE and then transferred to PVDF membranes (Millipore). After block with 5% BSA diluted in TBST, the membranes were probed overnight at 4 °C with the following primary antibodies: anti-TRPM7 (Abcam, ab729), anti-β-actin (Santa Cruz, sc-81178), anti-cleaved caspase-3 (Cell Signaling Technology, 9661), anti-p-MEK 1/2 (Santa Cruz, sc-81503), anti-MEK 1/2 (Santa Cruz, sc-81504), anti-p-ERK 1/2 (Santa Cruz, sc-81492), anti-ERK 1/2 (Santa Cruz, sc-292838). After wash with TBST, the membranes were further incubated with secondary antibodies conjugated with horseradish peroxidase (HRP). Protein bands were detected by chemiluminescence with the ECL detection reagent (Amersham Biosciences). The densitometric analysis of protein bands was performed by ImageJ software [48 (link)]. Results were normalized to those of β-actin.

NHS-Rhodamine, Opti-MEM, anti-uPA antibody, and TRIzol reagent were purchased from Thermo Fisher Scientific. DAPI was obtained from Dojindo. Fibrinogen was purchased from Sigma-Aldrich. RSK inhibitor SL0101 and MEK inhibitor PD98059 were obtained from Merck Millipore. Anti-phospho-RSK1 (Ser380), anti-RSK1, anti-RSK2, anti-GAPDH, anti-phospho-ERK1/2 (Thr202/Tyr204), and anti-phospho-MEK1/2 (Ser217/Ser221) antibodies were purchased from Cell Signaling Technology. Anti-ERK1/2, and anti-MEK1/2 antibodies were purchased from Santa Cruz Biotechnology. LentiCRISPRv2, pMD2.G, and psPAX2 were obtained from Addgene. FuGENE HD was purchased from Promega. PrimeScript RT reagent Kit and SYBR Premix Ex Taq II were obtained from TaKaRa.

Cells were lysed in SDS sample buffer composed of 1.5% dithiothreitol, 2% SDS, 80 mM Tris-HCl (pH 6.8), 10% glycerol and 0.01% bromophenol blue. The lysates were boiled in the buffer for 5 min and separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes. The membranes were blocked with bovine serum albumin or milk for 1 h and probed with use of primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Fisher Scientific). Proteins were visualized by enhanced chemiluminescence (Fisher Scientific) using the GE Imager 600 System. Antibody used were anti-Plk-1 (Millipore, Cat#05-844, Lot#2477015), anti-c-Abl (Cell Signaling, Cat#2862 S, Lot#13), anti-paxillin (BD Biosciences, Cat#610051, Lot#7208686), anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase. Ambion, Cat# AM4300, Lot#1311029), anti-MEK1/2 (Santa Cruz, Cat#Sc-436, Lot# H3011), anti-ERK1/2 (Cell Signaling, Cat#4695, Lot#8), anti-p-MEK1/2 (Santa Cruz, Cat# Sc-81503, Lot# I1813), anti-p-ERK1/2 (Cell Signaling, Cat# 9106 S, Lot# 38). Antibodies against phospho-vimentin (Ser-56) and total vimentin were custom made by Synpep Inc (CA, USA) and previously characterized^{18 (link),36 (link)}. The levels of proteins were quantified by scanning densitometry of immunoblots (Fuji Multigauge Software). The luminescent signals from all immunoblots were within the linear range.