A total of 84 prostate cancer tissues and 22 precancerous tissues were obtained from the Ningbo Pathology Center of Zhejiang Province. Each case took 2–3 slides for experimentation. Slides were deparaffinized and hydrated at room temperature. Immunohistochemical staining was performed by following the manufacturer's directions of a specific kit (Absin, China). 4E‐BP1 (dilution 1:100; Cell Signaling, US) and p4E‐BP1 (dilution 1:100; Cell Signaling, US) were applied as the primary antibody at 37℃ for 12 h. For 4EBP1 or p4EBP1 scoring, the cytoplasmic staining intensity (4E‐BP1 or p4E‐BP1 typically stained the cytoplasmic of cancer cells) was rated as negative (no staining at all), weak (1+ staining intensity), moderate (2+ staining intensity), or strong (3+ staining intensity). In addition, it was stipulated that the product of the area percentage score of positive cells and the cell staining intensity score is regarded as a comprehensive staining score, with a score of ≥3 as positive, and a score of 0–2 as negative.
4e bp1
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China
4E-BP1 is a protein that plays a key role in the regulation of translation initiation, a critical step in protein synthesis. It functions by binding to and inhibiting the activity of the eukaryotic translation initiation factor 4E (eIF4E), thereby suppressing the recruitment of ribosomes to mRNA and inhibiting the initiation of protein synthesis.
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Lab products found in correlation
P 4ebp1, by Cell Signaling Technology (124 mentions)
P70s6k, by Cell Signaling Technology (87 mentions)
P akt, by Cell Signaling Technology (59 mentions)
Phospho 4ebp1, by Cell Signaling Technology (52 mentions)
Gapdh, by Cell Signaling Technology (48 mentions)
β actin, by Cell Signaling Technology (44 mentions)
P mtor, by Cell Signaling Technology (41 mentions)
P p70s6k, by Cell Signaling Technology (39 mentions)
Cleaved caspase 3, by Cell Signaling Technology (34 mentions)
Eif4e, by Cell Signaling Technology (30 mentions)
β actin, by Merck Group (27 mentions)
Caspase 3, by Cell Signaling Technology (25 mentions)
Bcl 2, by Cell Signaling Technology (25 mentions)
Cyclin d1, by Cell Signaling Technology (23 mentions)
Phospho 4e bp1 thr37 46, by Cell Signaling Technology (22 mentions)
Pakt s473, by Cell Signaling Technology (21 mentions)
P akt ser473, by Cell Signaling Technology (20 mentions)
P 4e bp1 thr37 46, by Cell Signaling Technology (20 mentions)
Raptor, by Cell Signaling Technology (20 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (20 mentions)
409 protocols using 4e bp1
1
Immunohistochemical Analysis of 4E-BP1 and p4E-BP1 in Prostate Cancer
2
Protein Synthesis Signaling Pathway Analysis
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Protein content was compared across groups via Western Blot analysis using previously described methods [12 (link), 16 (link)]. 4-hydroxynonenal (4-HNE) conjugated proteins were measured as an index of oxidative damage (Abcam, Cambridge, MA; product #46546). To measure markers of protein synthesis signaling, membranes were incubated with antibodies against phosphorylated Akt (Ser 473), phosphorylated proline rich Akt substrate of 40kD (PRAS40) (Thr 246), phosphorylated mammalian target of rapamycin (mTOR) (Ser 2448), and phosphorylated 4E-binding protein 1 (4E-BP1) (Thr 37/46), all purchased from Cell Signaling Technology (Danvers, MA). Subsequently, membranes were stripped, blocked, and incubated with antibodies against total Akt, GSK3β, PRAS40, mTOR, ribosomal protein s6, and 4E-BP1 all purchased from Cell Signaling Technologies (Danvers, MA). For all western blots where both phosphorylated protein and total protein were measured, the data were analyzed as a ratio of the phosphorylated to total protein ratio by dividing the density of phosphorylated protein by the density of total protein. All western blots were normalized to α-tubulin as a loading control. Product numbers for specific antibodies from Cell Signaling Technologies are as follows: Akt (#2920), pAkt (#4058), PRAS40 (#2691), PRAS40 (#2997), p-mTOR (#5536), mTOR (#2983), 4E-BP1 (#9452), and p-4E-BP1 (#9459).
3
Western Blot Analysis of Signaling Proteins
4
Protein Extraction and Western Blot Analysis
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The proteins were extracted using standard techniques [40 (link)]. The antibodies for IRF-8, p-S6, S6, p-4EBP-1, 4EBP-1, and horseradish peroxidase-conjugated anti-rabbit IgG for Western blot analysis were procured from Cell Signaling Technology (MA, USA). The protein bands were visualized using enhanced chemiluminescence (ECL) plus Western blotting detection reagents (Millipore, MA, USA). In the present study, α-tubulin was used as an internal control.
5
Quantifying Liver Protein Levels in Tg(TXN2) Mice
6
Quantifying Liver Protein Levels in Tg(TXN2) Mice
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Levels of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) were measured in the total cell lysates from liver of young (4-6 months old) Tg(TXN2)+/0 and WT mice by Western blot analysis using mouse p70S6K1, phospho-p70S6K1, 4E-BP1, and phospho-4EBP1 antibodies (Cell Signaling Technology, Inc., Danvers, MA).
Total cell lysates from liver of young (4-6 months old)Tg(TXN2)+/0 and WT mice were prepared, and detection of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) was performed using Western blots. The amount of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) was quantified by a densitometer, and the data were expressed as the relative amount of protein in lysates using β-actin as an internal standard.
Total cell lysates from liver of young (4-6 months old) Tg(TXN2)+/0 and WT mice were prepared, and detection of HIF-1α (Catalog No. Ab82832; Abcam, Cambridge, MA) was performed using Western blots. The amount of HIF-1α was quantified by a densitometer, and the data were expressed as the relative amount of protein in lysates using β-actin as an internal standard.
Total cell lysates from liver of young (4-6 months old)Tg(TXN2)+/0 and WT mice were prepared, and detection of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) was performed using Western blots. The amount of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) was quantified by a densitometer, and the data were expressed as the relative amount of protein in lysates using β-actin as an internal standard.
Total cell lysates from liver of young (4-6 months old) Tg(TXN2)+/0 and WT mice were prepared, and detection of HIF-1α (Catalog No. Ab82832; Abcam, Cambridge, MA) was performed using Western blots. The amount of HIF-1α was quantified by a densitometer, and the data were expressed as the relative amount of protein in lysates using β-actin as an internal standard.
7
Protein Expression Analysis by Western Blot
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Cell and tissue extracts were used for western blots to detect P(S473)-Akt (#9271) 1:1000, P(T308)-Akt (#9275) 1:1000, Akt (#9272) 1:1000, P(T37/46)4E-BP1 (#9459) 1:1000, 4E-BP1 (#9452) 1:1000, P(S240-244)rpS6 (#2215) 1:1000, P(T202/Y204)-ERK1/2 (#9101) 1:1000, ERK1/2 (#9102) 1:1000 all from Cell Signaling, β-actin (A1978) 1:5000 from Sigma, Periostin (sc-67233) and rpS6 (sc-100832) 1:2000 from Santa Cruz Biotechnology, and GAPDH (MAB 374) from Millipore.
8
Protein Expression Analysis via Western Blot
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Total protein lysates were isolated in lysis buffer (20 mmol/L Tris base, 1% Triton X‐100, 50 mmol/L NaCl, 250 mmol/L sucrose, 50 mmol/L NaF, 5 mmol/L Na2P2O7, plus Halt™ protease and phosphatase inhibitor cocktail [Thermo Scientific]). Equal amounts of protein (100 μg) were loaded and resolved on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Blots were probed with antibodies against ACSL1 (catalog No. 4047), phosphorylated p70 (P‐p70) S6 kinase (S6K) (catalog No. 9234), phosphorylated 4E‐BP1 (catalog No. 2855), and phosphorylated AMP‐activated protein kinase (P‐AMPK) (catalog No. 2531), and were then stripped and reprobed with p70 S6K (catalog No. 9092), 4E‐BP1 (catalog No. 9644), or AMPKα (catalog No. 2532) antibodies, respectively (all antibodies from Cell Signaling). GAPDH (Abcam, catalog No. ab8245) was used as loading control.
9
Comprehensive Immunoblotting Assay Protocol
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Abs from the following sources were used for immunoblotting. From Cell Signaling Technologies, we used: 4EBP1 (9644); (p)-4EBP1 (2855); 4EBP2 (2845); AKT (4691); (p)-AKT Ser473 (4060); EIF4E (2067); EIF4G (2469); EIF4E (2067); ERK (4695); (p)-ERK (4370); FGFR4 (8562); FOXO1 (2880); HA (3724); P70S6K (2708); (p)-P70S6K (9205); (p)-p90RSK Thr359/Ser363 (9344); MTOR (2983); RAS (67648); RPS6 (2217); (p)-RPS6 Ser240/244 (2215); (p)-RPS6 Ser235/236 (2211); WDR59 (53385). ProteinTech: GAPDH (60004-1-Ig); MIOS (20826-1), SEC13 (15397-1-AP); WDR24 (20778-1). From Abcam, we used SEH1L (ab218531). From Santa Cruz Biotechnology, we used LAMP2 (sc-18822). And from Sigma, we used Actin (A2228) and FLAG (F1804). Small molecules, including ulixertinib, sapanisertib, trametinib, and temsirolimus, were purchased from SelleckChem, and on-target effects were validated by immunoblot. RMC-6272 was provided by Revolution Medicines. Sequences used for sgRNA and shRNA are listed in Supplemental Table 2 .
10
Western Blot Analysis of Cell Signaling
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Immunoblotting was performed using a standard protocol as described previously (35) . Cells were lysed in NP-40 buffer containing Protease and Phosphatase Inhibitors Cocktails (Millipore), 0.2 mmol/L phenylmethylsulfonyl fluoride, and 10 mmol/L N 0 ethylmalamide. Protein quantification was conducted using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, #23225). Proteins were resolved by 4%-20% gradient SDS-PAGE. Primary antibodies were procured from Cell Signaling Technology: UCHL1 (13179), ATF3 (33593), ATF4 (11845), pSer51 eIF2a (3398), cleaved caspase-3 (9664), pSer235/236 S6 Ribosomal Protein (4858), S6 Ribosomal Protein (2217), 4E-BP1 (9452), and pThr37/46 4E-BP1 (2855). Other antibodies included UCHL1 (MAB6007, R&D Systems), PSMA7 (PA5-22289, Invitrogen), APEH (376612, Santa Cruz Biotechnology), cleaved caspase-3 (AF835, R&D Systems), and actin-HRP (Sigma).
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