Cells were lysed in lysis buffer (25 mM Tris pH 8.0, 125 mM NaCl, 1 mM MgCl2, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1X protease inhibitor (Sigma-Aldrich), phosphatase inhibitor cocktails #2 and #3 (Sigma-Aldrich), and 1 mM PMSF). Cell lysates were resolved on a Nupage 4-12% Bis-Tris gradient gel (Life Technologies) and probed using antibodies recognising phospho- and total eIF4E1 (Cell Signaling #9741 and Santa Cruz sc-9976), phospho- and total ERK (#9101 and #9102, Cell Signaling), c-Myc (#9402), n-Myc (#9405), p-MNK1/2 (#2111), dicer (#3363), eIF4A (#2013) (Cell Signaling), GAPDH (ab8245), BTK (ab54219), YY1 (ab12132) (Abcam), eIF4E3 (Proteintech: N-terminal, #17282-1-AP), MNK1 (sc-6965), MNK2 (sc-6964), CDK6 (sc-7961), eIF4G (sc-11373), MCL-1 (#sc-819) (Santa Cruz) or GFP (EVN-AB513, Axxora). All primary antibodies were used at 1: 1,000 dilution. Densitometry analyses were performed using ImageJ software (NIH) and presented as ratio of target band signal intensity to GAPDH band signal intensity. Full immunoblots are presented in Supplementary Fig.10 .
Eif4a
Manufactured by Cell Signaling Technology
Sourced in United States, Germany, United Kingdom
EIF4A is a highly conserved ATP-dependent RNA helicase that is a core component of the eukaryotic translation initiation complex. It is responsible for unwinding secondary structures in the 5' untranslated region of mRNA, facilitating ribosome binding and translation initiation.
Automatically generated - may contain errors
Lab products found in correlation
Eif4g, by Cell Signaling Technology (5 mentions)
Eif4e, by Cell Signaling Technology (4 mentions)
Phosphatase inhibitor cocktails 2 and 3, by Merck Group (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
C myc, by Cell Signaling Technology (2 mentions)
Nupage 4 12 bis tris gradient gel, by Thermo Fisher Scientific (2 mentions)
N myc, by Cell Signaling Technology (2 mentions)
P mnk1 2, by Cell Signaling Technology (2 mentions)
Eif2α, by Cell Signaling Technology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Pdcd4, by Cell Signaling Technology (2 mentions)
P eif4b, by Cell Signaling Technology (2 mentions)
4e bp1, by Cell Signaling Technology (2 mentions)
Mcl 1, by Santa Cruz Biotechnology (1 mentions)
Eif4g, by Santa Cruz Biotechnology (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab21060, by Abcam (1 mentions)
Eif4gi, by Cell Signaling Technology (1 mentions)
Ab177939, by Abcam (1 mentions)
Species specific horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
11 protocols using eif4a
1
Immunoblot analysis of cellular signaling
2
Affinity Chromatography Protein Profiling
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Proteins in equal volumes of affinity chromatography effluents or proteins associated with 700 nM 40S ribosome were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes and probed with antibodies against eIF3d (Abcam, Ab155419), eIF4E (Cell Signaling, #9742), eIF4A (Cell Signaling, #2013), eIF2α (Cell Signaling, #9722), eIF4GI (Cell Signaling, #2858), PABP (Abcam, Ab21060), eIF1A (Abcam, Ab177939), PAIP-1 (Abcam, Ab175211) or ribosomal protein S6 (Cell Signaling, #2317). Membranes were then incubated with species-specific horseradish peroxidase-conjugated secondary antibody (Cell Signaling) followed by detection with ECL substrate (Pierce).
3
Western Blot Analysis of Translation Regulators
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Western blot was described previously [6 (link)]. Briefly, cells were lysed using Laemmli buffer containing 10% dithiothreitol (DTT, Thermo Fisher Scientific, Carlsbad, USA), and then denatured by heating at 95 °C for 5 min. Proteins were detected with antibodies against eIF4A (Cell Signaling Technology, Danvers, USA), eIF4E (Cell Signaling Technology), eIF4G (Cell Signaling Technology), PDCD4 (Cell Signaling Technology), and SA11 rotavirus VP4 (provided by professor Harry Greenberg, Stanford University School of Medicine, USA). The bound antibodies were visualized with Odyssey (LI-COR Biosciences, USA). The β-actin (Santa Cruz) was used as a loading control.
4
Cloning and Immunodetection of PC-1 and 4E-BP1
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The sequence of PC-1 was amplified from human PC-1 cDNA and the PCR products were sub-cloned into pGEX-5T, pEGFP-N1, and pCMV-2B-tag vector, respectively. The sequence of 4E-BP1 was amplified from human 4E-BP1 cDNA and the PCR products were sub-cloned into pGEX-5T, pET22b, pEGFP-N1, and pDsRed-N1 vetor, respectively. The plasmid encoding Myc-ubiquitin was a gift from Dr. Cheng Cao (Beijing Institute of Biotechnology, Beijing, China). The specific shRNA constructs targeting the PC-1 and control construct was obtained from Cenechem Company (Shanghai, China). For immunoprecipitation and western blotting, the following primary antibodies were used: polyclonal antibody against PC-1 N-terminal 46 amino acids residues made by our laboratory [27 (link)], β-actin (Santa Cruz Biotechnology, Inc, Dallas, TX); antibodies against 4E-BP1, p4E-BP1(T70), mTOR, pmTOR(S2481), pmTOR(S2448), EIF4A, pRb(S780) and LC3B (Cell Signaling Technology, Danvers, MA); monoclonal antibodies against His (Invitrogen, Carlsbad, CA), Flag (Sigma-Aldrich, St Louis, MO), GFP (Abmart, Berkeley, NJ), MYC, Ub, p21, and p27 (Santa Cruz Biotechnology, Inc, Dallas, TX). The secondary antibodies were either horseradish peroxidase-linked anti-rabbit or anti-mouse both from Zhongshan Golden Bridge Biotechnology.
5
Protein Extraction and Immunoblotting Protocol
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Cells were lysed in lysis buffer (25 mM Tris pH 8.0, 125 mM NaCl, 1 mM MgCl2, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1X protease inhibitor (Sigma-Aldrich), phosphatase inhibitor cocktails #2 and #3 (Sigma-Aldrich), and 1 mM PMSF). Cell lysates were resolved on a Nupage 4–12% Bis-Tris gradient gel (Life Technologies) and probed using antibodies recognizing phospho- and total eIF4E1 (Cell Signaling #9741 and Santa Cruz sc-9976), phospho- and total ERK (#9101 and #9102, Cell Signaling), c-Myc (#9402), n-Myc (#9405), p-MNK1/2 (#2111), dicer (#3363), eIF4A (#2013) (Cell Signaling), GAPDH (ab8245), BTK (ab54219), YY1 (ab12132) (Abcam), eIF4E3 (Proteintech: N-terminal, #17282-1-AP), MNK1 (sc-6965), MNK2 (sc-6964), CDK6 (sc-7961), eIF4G (sc-11373), MCL-1 (#sc-819) (Santa Cruz) or GFP (EVN-AB513, Axxora). All primary antibodies were used at 1:1,000 dilution. Densitometry analyses were performed using ImageJ software (NIH) and presented as ratio of target band signal intensity to GAPDH band signal intensity. Full immunoblots are presented in Supplementary Fig. 10 .
6
Detection of Transcription Factor and RBP
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c-Myb was detected with Myb-specific monoclonal antibodies 5E11 (35 (link)). Pdcd4 was detected with a rabbit antiserum against the N-terminus of human Pdcd4 (12 (link)). Antibodies against GFP (Roche Diagnostics, Mannheim, Germany), eIF4A, eIF4G, eIF4E, PABP (Cell Signalling Technology), Y14 (Santa Cruz Biotechnology) and the Flag, Myc and His-tag were obtained from commercial suppliers. Antiserum against Drosophila PABP was kindly provided by E. Izaurralde (Tübingen, Germany).
7
Immunoprecipitation of HA-tagged VEEV nsP3
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U87MG cells were infected with rTC-83-nsP3-HA at a multiplicity of infection (MOI) of 20. At 6 hours post infection (hpi) cells were collected and lysed in a buffer as previously described (Amaya et al., 2014 (link), Amaya et al., 2015 (link)). Two milligrams of total protein was utilized for immunoprecipitation using Dynabeads® protein G beads with antibodies to HA (Abcam, ab18181) or an isotype IgG control. The immunoprecipitates were washed 4× with TNE50 + 0.1% NP-40.
Preparation of whole cell lysates and western blot have been previously described (Amaya et al., 2014 (link)). Primary antibodies to HA, DDX1 (Abcam, ab31963), DDX3 (Cell Signaling, 2635), VEEV-capsid (BEI Resources, NR-9403), HRP conjugated actin, PABP (Santa Cruz Biotechnology, sc-28834), TIA-1 (Santa Cruz Biotechnology, sc-1751), eIF4G (Cell Signaling, C45A4) and eIF4A (Cell Signaling, C32B4) were used according to the manufacturer's instructions. The densitometric counts of the protein bands of interest were normalized to those of the actin bands and are illustrated graphically.
Preparation of whole cell lysates and western blot have been previously described (Amaya et al., 2014 (link)). Primary antibodies to HA, DDX1 (Abcam, ab31963), DDX3 (Cell Signaling, 2635), VEEV-capsid (BEI Resources, NR-9403), HRP conjugated actin, PABP (Santa Cruz Biotechnology, sc-28834), TIA-1 (Santa Cruz Biotechnology, sc-1751), eIF4G (Cell Signaling, C45A4) and eIF4A (Cell Signaling, C32B4) were used according to the manufacturer's instructions. The densitometric counts of the protein bands of interest were normalized to those of the actin bands and are illustrated graphically.
8
Western Blot Analysis of Apoptosis-Related Proteins
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Western blot analysis was performed as described previously (13 (link)). Briefly, GBC-SD and SGC-996 cells were washed twice with ice-cold PBS and then lysed with ice-cold RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) supplemented using EDTA-free protease inhibitor cocktail (cat. no. 11836170001; Roche Diagnostics), and followed by protein concentration determination with BCA assay. Lysates were kept on ice for 30 min and then centrifuged at 14,000 × g for 20 min at 4°C. Equal amounts (20 µg) of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were block with 5% bovine serum albumin (BSA) in Tris-buffered saline Tween-20 for 2 h and then incubated with primary antibodies at 4°C overnight. Immunoreactive proteins were detected with horseradish peroxidase-conjugated secondary antibodies. The primary antibodies used were: Caspase-8 (cat. no. 9746; Cell Signaling Technology, Inc.), caspase-3 (cat. no. 9662; Cell Signaling Technology, Inc.), cleaved-caspase-3 (cat. no. 9664; Cell Signaling Technology, Inc.), eIF4A (cat. no. ab31217; Abcam), c-FLIP (cat. no. ab8421; Abcam) and α-tubulin (cat. no. sc-5286; Santa Cruz Biotechnology, Inc.).
9
Western Blotting Protein Analysis
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Western blotting analyses were performed as described previously (Marabita et al., 2016 (link)). Antibodies for P-Akt, Akt, eIF4E, P-eIF4E, P-eIF4B, eIF4B, eIF4A, eIF4H, eIF4G, P-eIF4G, 4E-BP1, P-S6, S6 were taken from Cell Signaling. Actin was from Santa Cruz, and puromycin from Millipore. All quantifications of the western blots were done on at least four different blots for each protein, in each condition. Differences between groups were assessed using Student's t-test. Significance was defined as a value of P < 0.05 (95% confidence).
10
Modulation of THP-1 and MM-6 Differentiation
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Differentiation of THP-1 and MM-6 cells was performed using Phorbol 12-myristate 13-acetate (PMA; Merck Millipore) or 1-α, 25-dihydroxyvitamin D3 (VitD3; Sigma Aldrich). For the inhibition of kinases and proteases, the following inhibitors were applied: S6K1 inhibitor DG2 (S6K-I), PKR inhibitor (PKR-I; 6,8-Dihydro-8-1H-imidazol-5-ylmethylene)-7H-pyrrolo[2,3-g]benzothiazol-7-one), ALLN (N-[N-(N-Acetyl-L-leucyl)-L-leucyl]-L-norleucine), MG-132, Calpain-Inhibitor V (Merck Millipore), rapamycin (mTOR-I; Sigma Aldrich), UO126 (MEK-I; Cell Signaling, Danvers, USA), BI-D1870 (RSK-I; Axon Medchem BV, Groningen, Netherland), and Cathepsin-Inhibitor I (Applichem). For Western Blotting, specific antibodies directed against RSK, phosphorylated (p-)RSK (T573), ERK1/2, p-ERK1/2 (T202/Y204), rpS6, p-rpS6 (S335/236), eIF4B, p-eIF4B (S422), eIF4A, eIF2α, p-eIF2α (S51), PKR (Cell Signaling), p-PKR (T446; Abcam, Cambridge, UK), V5 Tag (Invitrogen), C/EBPβ, Calpastatin, Histon H4 (Santa Cruz), Actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or TATA box-binding protein (TBP; Sigma Aldrich) were used in combination with secondary horseradish peroxidase (HRP-)coupled antibodies (Vector Laboratories, Peterborough, UK). All media and reagents were of the best available grade and routinely tested for endotoxins with the Limulus Amoebocyte Lysate assay (Lonza).
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