The primary antibodies of TEAD1 (sc-376113), TEAD4 (sc-134071), and CTGF (L-20) (sc-14939) were from Santa Cruz (Dallas, TX, USA). YAP1 (ab52771) antibody and HA tag (ab18181) were achieved from Abcam (Cambridge, MA, USA). AMOTL1 (HPA001196) and Flag-tag (F3165) antibodies were obtained from Sigma-Aldrich (St. Louis, MO, USA). Other primary antibodies were from Cell Signaling (Danvers, MA, USA), including p21 (#2946), p27 (#2552), pRb (Ser807/811) (#9308), Erk (#9102), pErk (#9101), Myc (#2278), cyclin D1 (#2978), c-Myc (#9402), and GAPDH (#2118). Anti-Mouse IgG-HRP (Dako, Glostrup, Denmark, 00049039, 1:30,000) and anti-Rabbit IgG-HRP (Dako, 00028856, 1:10,000) were used for secondary antibodies. The related protocol was suggested before [13 (link)].
Flag tag f3165
Manufactured by Merck Group
Sourced in United States
The Flag-tag (F3165) is a protein tag that can be fused to a target protein for the purpose of detection and purification. It consists of a small, hydrophilic polypeptide sequence that can be recognized by specific antibodies. This product can be used in various research applications that require the identification and isolation of tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
P erk, by Cell Signaling Technology (1 mentions)
Anti mouse igg hrp, by Agilent Technologies (1 mentions)
Anti rabbit igg hrp, by Agilent Technologies (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Yap1 ab52771 antibody, by Abcam (1 mentions)
Amotl1 hpa001196, by Merck Group (1 mentions)
P rb ser807 811, by Cell Signaling Technology (1 mentions)
0.45 μm pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Ha tag 3f10, by Roche (1 mentions)
Ab9132, by Abcam (1 mentions)
Ab52866, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
P erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Necrostatin 1, by Selleck Chemicals (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Anti ki67 ab15580, by Abcam (1 mentions)
4 protocols using flag tag f3165
1
Antibody Sources for Cell Signaling
2
Protein Expression Analysis by Western Blot
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Cells were lysed in RIPA buffer supplemented with complete protease inhibitor cocktail mix. Equal amount of proteins were separated by SDS-PAGE and subsequently transferred to 0.45 μm PVDF membrane (Thermo Scientific). Antibodies used were CHOP (D46F1), ATF-4 (D4B8), PRMT1 (F339), PARP (#9542) from Cell Signaling Technology; CREB-2/ATF4 (C-20), ATF-3 (C-19), GAPDH (H-12), β-actin (AC-15) from Santa Cruz Biotechnology; HA tag (3F10) from Roche; FLAG tag (F3165) from Sigma; anti-rabbit and anti-mouse HRP from Dako; anti-rabbit IgG light chain HRP (ab99697) from Abcam. Protein expression was detected by ECL™ Western Blotting Prime Detection Reagent (GE Healthcare) and visualized using Fluorochem E (Westburg, Cell Biosciences) or autoradiography.
3
Western Blot Analysis of Nuclear Proteins
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Nuclear extracts or IP eluates from brain tissues or HEK293T cells were resolved on 5–12% Tris-Glycine gels and transferred to nitrocellulose. Membranes were incubated overnight in the following primary antibodies: MeCP2 (Men-8, M7443, Sigma), TOP2β (H-286, sc-13059, Santa Cruz), TOP1 (H-5, sc-271285, Santa Cruz), Myc tag (ab9132, Abcam), FLAG tag (F3165, Sigma), α-Tubulin (EP1332Y, ab52866, Abcam). Following washes, membranes were incubated with HRP-conjugated secondary antibodies and visualized by enhanced chemiluminescence (ECL, Cell Signaling Technology) or secondary antibodies conjugated to IRdye 800 and imaged with LiCOR Odyssey.
4
Screening Compound Library Inhibitor Assay
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The screened compound library (L1400) was a product of Selleck Chemicals (Houston, TX). The inhibitors, including Necrostatin-1, GSK’872, z-VAD-fmk, MG132, SB216763, PD98059, and LY294002, were purchased from Selleck Chemicals. Cell culture medium, Fetal Bovine Serum (FBS), and supplements were purchased from Invitrogen (Grand Island, NY). Primary antibodies against EGFR (#4267, 1:1000), ERK1/2 (#9102, 1:1000), Akt (#4691, 1:1000), p-EGFR-Tyr1068 (#3777, 1:1000), p-ERK1/2-Thr202/Tyr204 (#4370, 1:1000), p-Akt-Ser473 (#4060, 1:1000), PARP (#9532, 1:1000), cleaved-caspase 3 (#9664, 1:1000), Bax (#14796, 1:1000), VDAC1 (#4661, 1:2000), cytochrome C (#11940, 1:1000), Mcl-1 (#39224, 1:1000), ubiquitin (#3936, 1:1000), ubiquitin (#43124, 1:1000), α-Tubulin (#2125, 1:5000), GSK3β (#12456, 1:1000), and β-actin (#3700, 1:10000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Flag-tag (F3165, 1:5000) antibody was obtained from Sigma Aldrich (St. Louis, MO). Antibodies against FBW7 (ab109617, 1:1000) and FBW7 (ab187815, 1:1000) were obtained from Abcam (Cambridge, UK). Antibodies for immunohistochemistry staining (IHC), including anti-ki67 (ab15580, 1:300) and anti-p-Akt (ab81283, 1:100) were obtained from Abcam. Anti-p-EGFR (#3777, 1:200), and anti-Mcl-1 (#39224, 1:100) were purchased from Cell Signaling Technology, Inc.
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