Cell lysates were separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE). Primary antibodies used were: PRLR (D01PID5618, Abnova, Taiwan); Moesin (SC6410, Santa Cruz, Texas, USA); p-Moesin (SC12895, Santa Cruz, Texas, USA); FAK (SC271195, Santa Cruz, Texas, USA); p-FAK (SC11765-R, Santa Cruz, Texas, USA); c-Src (SC5266, Santa Cruz, Texas, USA); p-c-Src (SC166860, Santa Cruz, Texas, USA); GAPDH (SC59540, Santa Cruz, Texas, USA). The secondary antibodies Anti-rabbit (SC2357, Santa Cruz, Texas, USA) (1:3000) for PRLR; Anti-goat (SC2768, Santa Cruz, Texas, USA) (1:3000) for Moesin, p-Moesin and Actin, and Anti-Mouse (SC2005, Santa Cruz, Texas, USA (1:3500) for FAK, p-FAK, c-Src, p-c-Src, and GAPDH were incubated for 2 h in room temperature. Primary and secondary antibodies were incubated with the membranes, followed by three 5-min washings with TRIS-buffered saline-Tween 20. Immunodetection was carried out using enhanced chemiluminescence and was recorded with a quantitative digital imaging system (Quantity One, BioRad, Hercules, CA, USA), enabling us to assess saturation. Band densitometric analysis was quantified, as previously described (17 (link)), with the ImageJ image program (National Institute of Health – NIH, USA) using conditions ensuring analysis in the linear range of detection.
C src
Manufactured by Santa Cruz Biotechnology
Sourced in United States
C-Src is a protein tyrosine kinase that plays a crucial role in cellular signaling pathways. It is involved in the regulation of cell growth, differentiation, and survival. C-Src is a commonly used target for research in various fields, including cell biology, cancer biology, and signal transduction.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (5 mentions)
P c src, by Santa Cruz Biotechnology (5 mentions)
Gapdh, by Santa Cruz Biotechnology (4 mentions)
Nf κb p65, by Santa Cruz Biotechnology (3 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (3 mentions)
Bcl 2, by Santa Cruz Biotechnology (3 mentions)
Mmp 2, by Santa Cruz Biotechnology (3 mentions)
P erk, by Santa Cruz Biotechnology (3 mentions)
Pt183 y185 jnk, by Cell Signaling Technology (2 mentions)
Pt202 y204 erk, by Cell Signaling Technology (2 mentions)
Ps32 36 iκbα, by Cell Signaling Technology (2 mentions)
Ps176 180 ikkα β, by Cell Signaling Technology (2 mentions)
Py416 c src, by Cell Signaling Technology (2 mentions)
Pt180 y182 p38, by Promega (2 mentions)
Mouse trip6, by BD (2 mentions)
Traf6, by Santa Cruz Biotechnology (2 mentions)
Histone h3, by Cell Signaling Technology (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Cleaved caspase 3, by Santa Cruz Biotechnology (2 mentions)
P fak, by Santa Cruz Biotechnology (2 mentions)
27 protocols using c src
1
Protein Expression Analysis by Western Blot
2
Immunoblotting, Co-IP, and Recombinant Protein Purification
3
Curcumin-Lecithin Antioxidant Activity Study
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Curcuma Longa Linn (powdered form) and lecithin (L-α-phosphatidylcholine) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The organic solvents such as toluene and dichloromethane were purchased from Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from GE Healthcare (Logan, UT, USA). The following antibodies were obtained: c-Src, phospho-c-Src, PKC, phospho-PKC, JNK, phospho-JNK, p38, phospho-p38, ERK, phospho-ERK, IκBα, phospho-IκBα, NF-κBp65, phospho-NF-κBp65, Bcl-2, Bax, cleaved caspase-3, and β-actin antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA); The following reagents were obtained: N-acetylcysteine (NAC) (Tocris, KOMA Biotech, Seoul, Korea) and 5-(and-6)-chloromethyl-2′,7′- dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) (Invitrogen, Carlsbad, CA, USA). PP2, SP600125, Bisindolylmaleimide I, and Bay11-7082 were obtained from MedChemExpress (Monmouth Junction, NJ, USA). All other reagents did not show any critical cytotoxic effects by themselves.
4
Cell Lysis and Immunoblotting Methodology
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Cells were grown in 6-well culture plates and harvested at 80% confluence to prepare whole-cell lysates using modified RIPA buffer containing Triton X-100 [24 (link)]. The spheroids cultured in the 3D collagen type I environment were harvested as described in a previous report [28 (link)]. Primary antibodies used for immunoblotting were as follows: phospho-Y416 Src, (Cell Signaling, Boston, MA, USA); c-Src (Santa Cruz Biotechnology, Santa Cruz, CA, USA); FAK, phospho-Y397FAK (BD Bioscience, San Jose, CA, USA); anti-HA (BioLegend, San Diego, CA, USA).
5
Antibody and RNA Interference Protocol for Src and FAK
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Antibodies against the phosph- FAK (catalog number: sc-374668) and c-Src (catalog number: SC-12928-R), total protein of c-Src (catalog number: SC-5266) and FAK (catalog number: SC-1688), VEGF (catalog number: SC-7269), SphK1 (catalog number: sc-365401) and β-actin (catalog number: sc-47778) were bought from Santa Cruz Biotechnology (Dallas, TX, USA). Small interfering RNAs were obtained from Dharmacon™ (Lafayette, CO, USA). Control mimic and miRNA mimic were provided by Thermo Fisher Scientific (Waltham, MA, USA). The TaqMan® MicroRNA Reverse Transcription Kit, qPCR primers and detection probes were all obtained from Applied Biosystems (Waltham, MA, USA). The Src inhibitor (10 µM PP2, catalog number: P0042) was supplied by Sigma-Aldrich (St. Louis, MO, USA) and the FAK inhibitor (10 µM FAKi, catalog number: 324878) by Calbiochem (San Diego, CA, USA).
6
Western Blot Analysis of EMT Markers
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For western blot analysis cells were lysed using RIPA buffer containing PhosSTOP Phosphatase Inhibitor Cocktail (Roche). Protein concentration was determined by Bradford assay (Bio‐Rad). Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences). The following antibodies were used: E2F1 (KH‐95; 1:500), EGFR (1:250), FGFR1 (Flg C15; 1:1000), TGFBR1 (V22; 1:1000), TGFBR2 (L21; 1:1000), Vimentin (V9; 1:1500), ZEB1 (H‐102; 1:1000), Snail (H-130; 1:500), SMAD2/3 (FL-425; 1:1000), and c-SRC (1:500) from Santa Cruz, E‐Cadherin (1:1500), and NFKB1 (C22B4; 1:1500) from Cell Signaling; N-Cadherin (610921; 1:1500) and FN1 (1:1000) from BD Bioscience, Actin (Sigma; 1:4000) and their corresponding HRP‐conjugated secondary antibodies (Pierce; 1:2000). Detection of HRP activity was performed with the ChemiDoc TouchTM Imaging System (BioRad) using ECL Plus (Amersham) or Super Signal West Femto (Thermo Scientific) Western Blotting Detection Reagents (GE Healthcare). Uncropped pictures of the immunoblots are shown in Supplementary Figs 9 , 10 , and 11 .
7
Immunoblotting Characterization of Focal Adhesion Dynamics
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Cell lysates were separated by SDS-PAGE in 8–10% gels and transferred into polyvinylidene difluoride (PVDF) membranes. Antibodies used were p-FAKY397 (611807), FAK (610088), and Arp3 (612135) (BD Transduction Laboratories); actin (sc-1615), LHR (sc-25828), c-Src (sc-5266), paxillin (sc-31010), p-paxillinY118 (sc-365020), cortactin (sc-11408), p-cortactinY466 (sc-101611), N-WASP (sc-13139), Arp2 (sc-15389), and p-Tyr (sc-7020) (Santa Cruz Biotechnology); p-N-WASPS484/485 (ab1964) (Chemicon International); p-SrcY418 (ab4816) (Abcam); and p-Arp2T237 (orb155730) (Biorbyt). Primary and secondary antibodies were incubated with the membranes using standard techniques. Immunodetection was accomplished using enhanced chemiluminescence and recorded with a quantitative digital imaging system (ChemiDoc XRS with Image Lab, Bio-Rad).
8
Molecular Mechanisms of Mitochondrial Regulation
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Antibodies against VDAC1 (B-6), IKKα/β (H-470), IKKβ (H-4), ΙΚΚγ (FL-419, B-3), HSP60 (K-19 and N-20), IκBα (C-21), Bcl-2, Bcl-xL, Cyclophilin D, c-Src, Enhanced green fluorescence protein (EGFP), MK2, p-MK2, and goat IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against p-IKKα/β, p-IκBα, ANT2 (E2B9D), Bax, p-Src (Y416), p-JNK (T183/Y185), JNK1/2, p38, and p-p38 were from Cell Signaling Technology (Danvers, MA, USA). Anti-cytochrome c antibody was from BD Bioscience (San Jose, CA, USA). Antibodies to Prx III and β-actin were obtained from AbFrontier (Seoul, Korea). Anti-MFF1 antibody was purchased from Proteintech (Rosemont, IL, USA). Recombinant GST-fused p38 enzyme was purchased from R&D systems (Minneapolis, MN, USA).
Mouse monoclonal antibody against α-tubulin, hydrogen peroxide, glucose oxidase, diamide, cyclosporine A, cycloheximide, coomassie brilliant blue R250, and DuoLink in situ fluorescence reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). SB203580, MG132, bongkrekic acid, lactacystin, bafilomycin A1, and 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid) were purchased from Calbiochem (San Diego, CA, USA). Tetramethylrhodamine, ethyl ester (TMRE) was purchased from Invitrogen (Waltham, MA, USA). MK inhibitor IV was purchased from Cayman Chemical (Ann Arbor, MI, USA).
Mouse monoclonal antibody against α-tubulin, hydrogen peroxide, glucose oxidase, diamide, cyclosporine A, cycloheximide, coomassie brilliant blue R250, and DuoLink in situ fluorescence reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). SB203580, MG132, bongkrekic acid, lactacystin, bafilomycin A1, and 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid) were purchased from Calbiochem (San Diego, CA, USA). Tetramethylrhodamine, ethyl ester (TMRE) was purchased from Invitrogen (Waltham, MA, USA). MK inhibitor IV was purchased from Cayman Chemical (Ann Arbor, MI, USA).
9
Antibody-Based Regulation of Colorectal Cancer Metastasis
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The antibodies used in this study were anti-Arp3; C-myc, Bax, Bcl-xl, Cleaved Caspase3, FAK, Phos-FAK, Phos-PI3K, MMP2, MMP9, tissue inhibitor of metalloproteinases 1 (TIMP1), Paxillin, Phos-Paxillin, Erk, Phos-Erk (Cell Signaling Technology), c-Src, Phos-c-Src (Santa Cruz Biotechnology) and Rac1 (Millipore). DAPI was purchased from Sigma-Aldrich. Phalloidin was provided by Invitrogen. All of the media were obtained from Gibco. All CRC cell lines were obtained from the American Type Culture Collection (ATCC). Medium was supplemented with 10% FBS, penicillin (100 units/ml) and streptomycin (100 units/ml). BALB/c mice and nude mice were from the National Rodent Laboratory Animal Resources, Shanghai Branch of China. All animal experimental protocols were approved by the Animal Investigation Committee of the Institute of Biomedical Sciences, East China Normal University.
10
Western Blot Analysis of Signaling Pathways
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The secondary antimouse and anti‐rabbit immunoglobulin G (IgG)–conjugated horseradish peroxidases (HRP) and primary antibodies against TSP‐2 (Cat. No. sc‐136238), MMP‐9 (Cat. No. sc‐393859), PLCβ (Cat. No. sc‐136040), PKCα (Cat. No. sc‐8393), c‐Src (Cat. No. sc‐8056), IKKα/β (Cat. No. sc‐7607), IκBα (Cat. No. sc‐1643) and p65 (Cat. No. sc‐8008) were purchased from Santa Cruz Biotechnology. p‐PLCβser537 (Cat. No. #2481), p‐PKCα/βThr638/641 (Cat. No. #9375), p‐IKKα/βSer176/180 (Cat. No. #2697), p‐IκBαSer32/36 (Cat. No. #9246), p‐c‐Srcser17 (Cat. No. #5473) and p‐p65Ser536 (Cat. No. #3033) were purchased from Cell Signaling Technology.
Dominant‐negative (DN) mutants of IKKα and IKKβ were kindly provided by Dr H. Nakano (Juntendo University, Tokyo, Japan). The human TSP‐2 recombinant protein was obtained from R&D Systems. The short interfering RNA (shRNA) plasmids were obtained from the National RNAi Core Facility Platform (RNAi Core, Academia Sinica). The inhibitors for PLCβ, PKCα, c‐Src and NF‐κB were obtained from Santa Cruz Biotechnology. All the other chemicals used in molecular biology were obtained from Sigma‐Aldrich.
Dominant‐negative (DN) mutants of IKKα and IKKβ were kindly provided by Dr H. Nakano (Juntendo University, Tokyo, Japan). The human TSP‐2 recombinant protein was obtained from R&D Systems. The short interfering RNA (shRNA) plasmids were obtained from the National RNAi Core Facility Platform (RNAi Core, Academia Sinica). The inhibitors for PLCβ, PKCα, c‐Src and NF‐κB were obtained from Santa Cruz Biotechnology. All the other chemicals used in molecular biology were obtained from Sigma‐Aldrich.
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