Western blot analysis was performed as previously described, with slight modifications [11 (link)]. Briefly, HUVEC lysates were lysed using RIPA buffer supplemented with protease inhibitor (Cell Signaling 5871S). Proteins were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel, transferred to a PVDF membrane (GE Healthcare, RPN303F), and blocked in 5% bovine serum albumin (BSA) in PBS with 1% tween-20 (Sigma P2287) for 1h at RT. Primary antibodies used were: rabbit anti-phospho-Smad1/5 (1:1000, Cell Signaling 9516), rabbit anti-Akt (1:1000, Cell Signaling 9272), rabbit anti-phospho-Akt (Ser473) (1:1000, Cell Signaling 4060), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) (1:1000, Cell Signaling 4370), rabbit anti-ERK 1/2 (1:1000, Cell Signaling 4695), mouse anti-HIF1α (1:500, Novus biologicals NB100-105), mouse anti-p53 (1:1000, Abcam ab1101) and rabbit anti-p53 (1:500, Abcam ab131442). Membranes were incubated with primary antibodies diluted in 1% BSA overnight at 4°C. Signal was detected with horseradish peroxidase (HRP) anti-rabbit (1:5000, Invitrogen G-21234) or HRP anti-mouse (1:30,000, Invitrogen 81–6720), and imaged via Clarity Western ECL Substrate (Bio-Rad 170–5061). Full original blots are shown (S6 Fig ).
Rabbit anti phospho erk1 2 thr202 tyr204
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, United States
Rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) is a primary antibody that detects ERK1/2 proteins phosphorylated at specific threonine and tyrosine residues. It is used to identify and quantify the levels of activated ERK1/2 in cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti erk1 2, by Cell Signaling Technology (3 mentions)
Rabbit anti akt, by Cell Signaling Technology (2 mentions)
Rabbit anti phospho akt ser473, by Cell Signaling Technology (2 mentions)
G 21234, by Thermo Fisher Scientific (1 mentions)
Ab1101, by Abcam (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Ab131442, by Abcam (1 mentions)
Rpn303f, by GE Healthcare (1 mentions)
Rabbit anti phospho smad1 5, by Cell Signaling Technology (1 mentions)
Nb100 105, by Abcam (1 mentions)
Fusion fx, by Vilber (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Luminata forte, by Merck Group (1 mentions)
Rabbit anti phospho p38 mapk thr180 tyr182, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg hrp, by Thermo Fisher Scientific (1 mentions)
Rabbit anti p38 mapk, by Cell Signaling Technology (1 mentions)
Erk1 2 137f5, by Cell Signaling Technology (1 mentions)
Rabbit anti p38 mapk antibody, by Santa Cruz Biotechnology (1 mentions)
Western lightning chemiluminescent kit, by PerkinElmer (1 mentions)
Trans blot transfer medium, by Bio-Rad (1 mentions)
7 protocols using rabbit anti phospho erk1 2 thr202 tyr204
1
Western Blot Analysis of HUVEC Lysates
2
Western Blot Analysis of MAPK Signaling
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P815-HTR cells were lysed in radio-immunoprecipitation assay buffer (RIPA) buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM Na3VO4, 5 mM NaF, and a protease inhibitor cocktail). A total of 30 µg of protein was separated per lane on 10% SDS-polyacrylamide gels, followed by transfer onto PVDF membranes (Millipore). The membranes were blocked in TBST (Tris-buffered saline containing 0.1% Tween-20) supplemented with 5% nonfat milk. Probing with the primary antibody was overnight at 4 °C; the blot was washed three times with TBST, and then incubated with appropriate secondary antibody (anti-rabbit HRP). After washing the blots three times in TBST, the protein bands were detected using a Western HRP substrate ECL kit (Luminata Forte, Millipore) and chemiluminescence imaging (Fusion FX, Vilber Lourmat). Primary antibodies were rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling #9211; 1: 1000), rabbit anti-p38 MAPK (Cell Signaling #9212; 1: 1000), rabbit anti-phospho-Erk1/2 (Thr202/Tyr204) (Cell signaling #9101; 1: 1000), and rabbit anti-Erk1/2 (Cell Signaling #9102; 1: 1000). Secondary antibody was goat anti-rabbit IgG HRP (Thermo Fisher; 1: 5000).
3
Measurement of Protein Levels in Cells
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Proteins were measured as previously described [4 (link), 6 (link), 9 (link)] using mouse anti-CBS (A-2) antibody, a mouse anti-CSE (30.7) antibody, mouse anti-MPST (H-11), rabbit anti-p38 MAPK antibody and rabbit anti–phospho–p38 MAPK (Thr180/Tyr182) antibody (all from Santa Cruz Biotechnology, Middlesex, UK) and, rabbit anti–extracellular signal–regulated kinase (ERK)–1/2 (137F5) and rabbit anti–phospho–ERK-1/2 (Thr202/Tyr204; purchased from Cell Signalling Technology, Ely, Cambridgeshire, UK).
4
Western Blot Analysis of Signaling Proteins
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Equal amounts of proteins were resolved in either 8 or 10% SDS-PAGE and electroblotted onto polyvinylidine difluoride membrane (PVDF) using semi-dry blotting system (Trans-Blot Transfer medium; Bio-Rad Laboratories). Membranes were blocked in 5% BSA in PBS containing 0.1% Tween-20 and incubated overnight at 4°C with antibodies against rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) (1:1000), rabbit anti-ERK1/2 (1:1000), rabbit anti-phospho-AKT (Ser473) (1:500), rabbit anti-AKT (1:1000) from Cell Signaling Technology (Danvers, MA); mouse anti-EGFR (1:200) and anti-pan actin (1:50 000) antibodies from Neomarker (Fremont, CA). After several washes, membrane was incubated with either goat anti-rabbit or goat anti-mouse horseradish peroxidase conjugated secondary antibodies (DakoCytomation, Denmark) (1:10 000). Specific protein bands were visualized with an enhanced chemiluminescence using Western Lightning chemiluminescent kit (Perkin-Elmer, MA).
5
Antibody and Knockdown Assay Protocol
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The following antibodies were used: rabbit anti-eIF4E2 (4EHP) (Genetex, GTX103977), mouse anti-eIF4E (BD Biosciences, 610270), rabbit anti-eIF4ENIF1 (4E-T; abcam, ab55881), rabbit anti-DDX6 (Bethyl Laboratories, A300-460A), rabbit anti-CNOT1 (Proteintech, 14276–1-AP), mouse anti-α-Tubulin (Santa Cruz, sc-23948), mouse anti-β-actin (Sigma, A5441), mouse anti-Flag (Sigma, F3165), rabbit anti-HA (Sigma, H6908), mouse anti-V5 tag (Invitrogen, R960-25), rabbit anti-PARP (Cell Signaling Cat# 9532S), rabbit anti-DUSP6 (abcam Cat# ab76310), rabbit anti-DUSP7 (abcam Cat# ab100921),), rabbit anti-CNOT9 (RQCD1) (Proteintech Cat# 22503–1-AP), mouse GAPDH (Santa Cruz, sc-32233), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Cat#4370), mouse anti-MEK1/2 (Cell Signaling Cat# 4694S), rabbit anti-phospho-MEK1/2 (Ser217/221; Cell Signaling Cat# 9121S), rabbit anti-phospho-RPS6 (Ser240/244) (Cell Signaling Cat# 2215), and mouse anti-RPS6 (C-8).
The following siRNA and shRNAs were used: ON-TARGETplus Non-targeting Control Pool (Dharmacon, D-001810-10-05), 4EHP siRNA SMARTpool (Dharmacon, L-019870–01), eIF4ENIF1 (4E-T) siRNA SMARTpool (Dharmacon, L-013237–01), CNOT1 siRNA SMARTpool (Dharmacon, L-015369–01-0005), CNOT9 siRNA SMARTpool (Dharmacon, L-019972–00), Non-Targeting shRNA Controls (Sigma, SHC002), and EIF4E2 shRNA (Sigma, TRCN0000152006).
The following siRNA and shRNAs were used: ON-TARGETplus Non-targeting Control Pool (Dharmacon, D-001810-10-05), 4EHP siRNA SMARTpool (Dharmacon, L-019870–01), eIF4ENIF1 (4E-T) siRNA SMARTpool (Dharmacon, L-013237–01), CNOT1 siRNA SMARTpool (Dharmacon, L-015369–01-0005), CNOT9 siRNA SMARTpool (Dharmacon, L-019972–00), Non-Targeting shRNA Controls (Sigma, SHC002), and EIF4E2 shRNA (Sigma, TRCN0000152006).
6
Comprehensive Protein Detection Reagents
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T-BSA (cat#: T3392) and fluorescein isothiocyanate-conjugated T-BSA(T-BSA-FITC) (cat#: T5771) were purchased from Sigma-Aldrich (MO, USA). BSA-FITC (cat#: SF063) was purchased from Solarbio (China). Actinomycin D (Act D; cat#: HY-17559) and Cycloheximide (CHX; cat#: HY-12320) were purchased from MedChemExpress (NJ, USA). Flutamide (F; cat#: F0663) and Dimethyl sulfoxide (DMSO; cat#: D0798) were purchased from Tokyo Chemical Industry Co., Ltd (Japan). SCH772984 (cat#: S7101) was purchased from Selleck Chemicals (TX, USA). Rabbit anti-PSD95 (cat#: ab18258), rabbit anti-phospho-eIF4E (S209) (cat#: 76256), rabbit anti-eIF4E (total) (cat#: 33766), rabbit anti-GAPDH (cat#: ab9485) antibodies were purchased from Abcam (MA, USA). Rabbit anti-phospho-Erk1/2 (Thr202/Tyr204) (cat#: 9101) and mouse anti-Erk1/2 (total) (cat#: 9107) antibodies were purchased from cell signaling technology (MA, USA). Rabbit anti-ZIP9 antibody (cat#: GTX31817) was purchased from GeneTex (CA, USA). Mouse anti-Gnα11 (cat#: sc-390382) and normal mouse IgG (cat#: sc-2025) antibodies were purchased from Santa Cruz Biotechnology (MA, USA).
7
Immunoblotting Analysis of Aortic Tissue
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Frozen sections of descending aorta or aortic sinus (base of the heart) were cut at a thickness of 10 µm, air-dried for 5 minutes and Immunoblotting. Aortic biopsies were lysed in ice cold RIPA buffer (Sigma) supplemented with 1mM dithiothreitol, phosphatase and protease inhibitor cocktail (PhosSTOP and cOmplete protease inhibitor cocktail; Roche). Western blots were performed using a standard protocol. Proteins were transferred by semi-dry transfer onto PVDF membrane. After blocking, membranes were probed with primary antibodies overnight. Antibodies used were Rabbit antiphospho-Akt (S473) (1:500; Cell Signalling Technology, 4058), Rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) (1:1000; Cell Signalling Technology, 9101), Rabbit anti-eIF4E (1:1000; Cell Signalling Technology, 2067), Rabbit anti-β-actin (1:1000; Abcam, ab8227). HRP-conjugated secondary antibodies were from GE Healthcare (rabbit, NA934) and R&D (sheep, HAF016).
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