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Pmek1 2 s221

Manufactured by Cell Signaling Technology
Sourced in United States

The pMEK1/2 (S221) product is a phospho-specific antibody that detects MEK1/2 proteins phosphorylated at Serine 221. This antibody can be used to measure the phosphorylation status of MEK1/2 proteins, which are key components of the mitogen-activated protein kinase (MAPK) signaling pathway.

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4 protocols using pmek1 2 s221

1

Western Blot Analysis of AXL Signaling

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Whole-cell extracts were obtained by lysing subconfluent proliferating cells or tissue samples from xenografts in radioimmunoprecipitation assay (RIPA) buffer. Standard western blot protocols were used [24 (link)]. After transferring to polyvinylidene difluoride membranes, the membranes were probed for specific proteins with enhanced chemiluminescence western blotting detection (GE Healthcare, Marlborough, MA) to probe. For GAS6 stimulation experiments, cells were serum-starved in 1% FBS-DMEM overnight and then stimulated for 30 min with 800 ng/mL recombinant GAS6. Antibodies against AXL, phosphorylated AXL (pAXL; Y702), AKT, phosphorylated AKT (pAKT; S473), ERK, phosphorylated ERK (pERK; T202/T204), MEK1/2, and phosphorylated MEK1/2 (pMEK1/2; S221), were obtained from Cell Signaling Technology (Danvers, MA). An anti–β-actin–horseradish peroxidase antibody was obtained from Santa Cruz Biotechnology (Dallas, TX).
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2

Antibody Generation and Immunoblotting Protocol

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Rabbit anti-TNS1 antibody was generated against human TNS1 aa1328–1339 peptide16 (link). Antibodies against E-cadherin (#9121), pMek1/2(S221) (#2338), pMek1/2(S217/221) (#9154), Mek1/2 (#9122), pERK1/2(T202Y204) (#9101), ERK1/2 (#9194), pAkt(S473) (#9271), Akt (#9272), pGSK3β(S9) (#5558), and GSK3β (#12456) were from Cell Signaling Technology. Anti-GAPDH (#CB1001) was from Millipore. Anti-Vinculin (#693291) was from MP Biomedical. Anti-mouse or rabbit IgG HRP conjugated secondary antibodies (#7076, #7074) were from Cell Signaling Technology. Alexa Fluor secondary antibodies (488/594) and Alexa Fluor 488/594 Phalloidin were from Thermo Scientific and anti-GP135/Podocalyxin (#3F2/D8) was from the Developmental Studies Hybridoma Bank at the University of Iowa. VECTASHIELD antifade DAPI mounting solution was from Vector Labs. Mek inhibitors CI-1040 (#S1020), selumetinib (#S1008), trametinib (#S2673) were from Selleckchem, and PD98059 (#9900), U0126 (#9903) were from Cell Signaling Technology.
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3

Western Blot Analysis of AXL Signaling

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Whole-cell extracts were obtained by lysing subconfluent proliferating cells or tissue samples from xenografts in radioimmunoprecipitation assay (RIPA) buffer. Standard western blot protocols were used [24 (link)]. After transferring to polyvinylidene difluoride membranes, the membranes were probed for specific proteins with enhanced chemiluminescence western blotting detection (GE Healthcare, Marlborough, MA) to probe. For GAS6 stimulation experiments, cells were serum-starved in 1% FBS-DMEM overnight and then stimulated for 30 min with 800 ng/mL recombinant GAS6. Antibodies against AXL, phosphorylated AXL (pAXL; Y702), AKT, phosphorylated AKT (pAKT; S473), ERK, phosphorylated ERK (pERK; T202/T204), MEK1/2, and phosphorylated MEK1/2 (pMEK1/2; S221), were obtained from Cell Signaling Technology (Danvers, MA). An anti–β-actin–horseradish peroxidase antibody was obtained from Santa Cruz Biotechnology (Dallas, TX).
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4

Antibody Validation for Signaling Pathways

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The β-actin (1:1000, sc-130656) and BCKDK (E-12) (1:1000, sc-374425) antibodies were purchased from Santa Cruz Technology, Inc. (Santa Cruz, CA, USA). Flag antibody (1:1000, #F1804, #F7425) was purchased from Sigma-Aldrich (St. Louis, MO, USA). His Mouse monoclonal antibody (1:1000, 4E6) was obtained from Pregene, Inc (Beijing, China). All the following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): p-BCKDHA (S293) (1:500, #40368), BCKDHA (1:500, #90198), MEK1/2 (1:1000, #8727), p-MEK1/2 (S221) (1:1000, #2338), p-ERK1/2 (T202/Y204) (1:2000, #4370), ERK1/2 (1:2000, #4695), p-RPS6 (S235/236) (1:1000, #2211), p-RPS6 (S240/244) (1:1000, #2215), RPS6(1:1000, #2217), E-cadherin (1:500, #3195), N-cadherin (1:500, #13116), and Vimentin (1:500, #5741). p-BCKDK (Y246) antibody was prepared by Abiocode, Inc (shanghai, China). Rabbit (1:3000, #E030120) and mouse (1:3000, #E030110) antibodies were obtained from EarthOx Life Sciences (San Francisco, CA, USA). We purchased BT2 (Cat No. ZC-26488) from ZZBIO. CO.LTD (Shang Hai, China). Dasatinib (Cat No. S1021) was purchased from Selleckchem, Inc (Houston, TX, USA).
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