Samples were collected in RIPA buffer (Sigma) containing Complete Protease Inhibitor Cocktail (Roche Diagnostics), and protein concentration was determined by Bio-Rad Protein Assay. Western blot analysis was performed using the following antibodies: KRAS (sc-30), Pan-RAS (sc-32), ERK1 (sc-271270), and c-Myc (sc-40) from Santa Cruz Biotechnology; Sox2 (#2748, #3579), Oct-4 (#2750), Nanog (#4893), Slug (#9585), Snail (#3879), phospho-ERK1/2 (#9101), RAS (#3965), MEK1/2 (Ser217/221) (#4694), phospho-MEK1/2 (Ser217/221) (#9121, #9154), CD44 (#3578, #3570), E-cadherin (#14472), vimentin (#3932) and cleaved caspase-3 (#9661) from Cell Signaling; N-cadherin (BD610920) and E-cadherin (BD610181) from BD Biosciences; Zeb1 (NBP-1-05987) from Novus Biologicals; and β-actin from Sigma.
C myc sc 40
Manufactured by Santa Cruz Biotechnology
Sourced in United States
C-Myc (sc-40) is a primary antibody product offered by Santa Cruz Biotechnology. It is a mouse monoclonal antibody that specifically recognizes the c-Myc protein.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (7 mentions)
Nanog, by Cell Signaling Technology (5 mentions)
Cleaved caspase 3, by Cell Signaling Technology (5 mentions)
Ripa buffer, by Merck Group (5 mentions)
Complete protease inhibitor cocktail, by Roche (5 mentions)
Zeb1 nbp 1 05987, by Novus Biologicals (3 mentions)
Phospho erk1 2, by Cell Signaling Technology (3 mentions)
Snail, by Cell Signaling Technology (3 mentions)
E cadherin, by Cell Signaling Technology (3 mentions)
Vimentin, by Cell Signaling Technology (3 mentions)
E cadherin, by BD (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Erk1 sc 271270, by Santa Cruz Biotechnology (2 mentions)
N cadherin, by BD (2 mentions)
Ab124822, by Abcam (2 mentions)
Ab168338, by Abcam (2 mentions)
Ab168381, by Abcam (2 mentions)
Gfp sc 9996, by Santa Cruz Biotechnology (2 mentions)
α synuclein ab27766, by Abcam (2 mentions)
α synuclein ab138501, by Abcam (2 mentions)
27 protocols using c myc sc 40
1
Western Blot Analysis of Stemness and EMT Markers
2
Immunofluorescence Characterization of Spheroid Cells
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Spheroid cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Following cell fixation, cells were incubated with antibodies for CD133 (MBS462020; Miltenyi Biotec), Nanog (#8822; Cell Signaling), Oct4 (#83932; Cell Signaling), Sox2 (#3570; Cell signaling), c-Myc (sc-40; Santa Cruz), N-cadherin (BD610920; BD Biosciences), and/or Slug (#9585; Cell Signaling) in a solution of PBS with 1% BSA and 0.1% Triton X-100 at 4 °C overnight. Cells were then stained with Alexa Fluor 488 (A-11029; ThermoFisher), Alexa Fluor 568 (A-11061; ThermoFisher), Alexa Fluor Plus 555 (A-32732; ThermoFisher, and Alexa Fluor 647 (A-21236; ThermoFisher). Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI, 28718-90-3; Sigma-Aldrich). Stained cells were visualized with an inverted confocal microscope (Leica Microsystems). Image processing was performed using Imaris, Version 7.6 (Bitplane).
3
Protein Phosphatase and Protease Inhibitor Cocktails
4
Comprehensive Protein Detection in Biological Samples
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Samples were collected in RIPA buffer (Sigma) containing Complete Protease Inhibitor Cocktail (Roche Diagnostics), and protein concentration was determined using the Bio-Rad Protein Assay. Western blots employed the following antibodies: CDK5RAP3 (sc-271776), ERK1 (sc-271270), Kras (sc-30) and c-Myc (sc-40) purchased from Santa Cruz Biotechnology; Sox2 (#2748, #3579), Oct4 (#2750), Nanog (#4893), Slug (#9585), Snail (#3879), phospho-ERK1/2 (#9101), phospho-JNK1/2 (#9251), phospho-p38 (#9211), Ras (#3965), CD44 (#3578, #3570), E-cadherin (#14472), vimentin (#3932) and cleaved caspase-3 (#9661) from Cell Signaling; N-cadherin (BD610920) and E-cadherin (BD610181) from BD Biosciences; Zeb1 (NBP-1-05987) from Novus Biologicals; and β-actin from Sigma.
5
Antibody Characterization for Helicase Enzymes
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The antibodies used in this study were as follows: USP28 (17707-1-AP, 1:1000 WB, Proteintech); RECQL1 (A300-450A, 1:1000 WB, Bethyl Lab, Montgomery); WRN (4666S,1:1000 WB,Cell Signaling); BLM (A300-110A,1:1000 WB, Bethyl Lab); RECQL4 (17008-1-AP,1:1000 WB, Proteintech); RECQL5 (A302-520A, 1:2000 WB, Bethyl Lab); Flag (F3165, 1:2000, Sigma); Ub (SC-8017, 1:200 WB, Santa Cruz Biotechnology); γH2AX (05-636, 1:500 IF, Millipore); Claspin (2800S, 1:1000 WB, Cell Signaling); c-MYC (SC-40, 1:200 WB, Santa Cruz); Phospho-Chk1-Ser345 (2348, 1:1000 WB, Cell Signaling); CHK1 (ab32531, 1:1000 WB, Abcam); GAPDH (60004-1-1g, 1:5000 WB, Proteintech); Tubulin (66240-1-1g, 1:5000 WB, Proteintech); ACTIN (66009-1-1g, 1:3000 WB, Proteintech). The secondary antibodies conjugated to horseradish peroxidase for Western blot and the secondary antibodies anti-mouse, -goat or -rabbit Alexa fluor 488 or 594 for immunofluorescence staining were purchased from Jackson ImmunoResearch Laboratories. siRNAs were synthesized by GenePharma.
6
Western Blot Analysis of Stem Cell and EMT Markers
7
Antibody panel for mitochondrial proteome
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Protein phosphatase inhibitor and protease inhibitor cocktails were purchased from Sigma–Aldrich. Antibodies for ClpX (ab168338, 1:2000), ClpP (ab124822, 1:1000), VDAC (ab34726, 1:1000), α-Synuclein (ab27766, 1:1000), α-Synuclein (ab138501, 1:3000) and α-Synuclein phosphor S129 (ab168381, 1:1000) were from Abcam. GFP (sc-9996, 1:1000), c-Myc (sc-40, 1:1000), α-Synuclein (sc-12767, 1:1000), Enolase (sc-15343, 1:1000) and HSP60 (sc-13115, 1:2000) were from Santa Cruz Biotechnology. β-Actin (A1978, 1:10000) was from Sigma–Aldrich. TH (MAB318, 1:1000) was from Millipore. ClpP (GTX115070, 1:200) was from Genetex. ClpP (NBP1-89557, 1:200) was from Novus. LonP (15440-1-AP, 1:2000), ERAL1 (11478-1-AP, 1:2000), CHCHD3 (25624-1-AP, 1:1000) and WFS1 (11558-1-AP, 1:1000) were from Proteintech. SOD2 (611580, 1:1000) was from BD bioscience. MFN1 (H00055669-M04, 1:1000) was from Abnova. Sirt3 (5490S, 1:1000) was from cell signaling technology.
8
Immunoblotting Assay for Protein Expression Analysis
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Cells were scraped, washed with 1 × PBS and lysed in lysis buffer. Protein concentrations were estimated using Bradford reagent (Biorad). Equal amount of protein was loaded for immunoblotting. Following SDS-PAGE, resolved proteins were electroblotted on PVDF membrane (Biorad). The membrane was blocked overnight in PBS containing 0.1% Tween-20 (PBST) and 1 % BSA. The membrane was then probed with primary antibody in PBST for 2 h at RT or overnight at 4 °C followed by three 10 min PBST washes at room temperature. Incubation with the secondary antibody was done for 1 h, followed by three 10 min PBST washes prior to chemiluminescence detection using ECL substrate (Thermo Scientific). Following are the antibodies used for the immunoblotting: Anti-MYCBP2 polyclonal rabbit antibody (ab86078, Abcam), cleaved caspase-3 (9661 S, Cell Signalling Technology), c-myc (sc-40 Santa Cruz Biotechnology), and tubulin (T6074, Sigma-Aldrich) were used at a 1:200 dilution.
9
Polyamine Pathway Inhibitors and Regulation
10
Western Blot Analysis of Cellular Proteins
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Total protein from cells was extracted with RIPA lysis buffer, quantified by a BCA kit (23225, Thermo Fisher Scientific), and subjected to 10% SDS-PAGE under denaturing conditions. The samples were transferred to nitrocellulose filter membranes (Merck Millipore, Burlington, MA, USA). Membranes were blocked for 1 hour with 5% milk PBS Tween-20 (PBST) buffer. Afterwards, membranes were incubated overnight at 4°C with primary antibodies against PRL-3 (generated by our laboratory).13 (link) RAP1 (A300-306A-2, Bethyl Laboratories, Montgomery, TX, USA), GAPDH (ab8245, Abcam, Cambridge, UK), PGC-1α (#2178, Cell Signaling Technology, Danvers, MA, USA), and c-Myc (sc-40, Santa Cruz Biotechnology, Dallas, TX, USA). Then, membranes were washed three times with PBST buffer, followed by incubation in goat anti-rabbit or anti-mouse secondary antibody conjugated with horseradish peroxidase for 1 hour at room temperature. After washing with PBST three times, protein bands were visualized using an eECL WB kit (CWBIO, Beijing, China).
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