Cells were homogenized in lysis buffer (Beyotime) and protein concentrations were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of total protein were separated by SDS-PAGE and transferred to pure nitrocellulose membranes. The bands were visualized using enhanced chemiluminescence (Millipore, Billerica, MA, USA). Digital images of the positive bands were analyzed with Quantity One software (Bio-Rad, Hercules, CA, USA). Antibodies targeting the following proteins were used: GAPDH (G9545, Sigma-Aldrich), MICAL-L2 (PA5-24826, Thermo Fisher Scientific), c-Myc (#13877, Cell Signaling Technology, Danvers, MA, USA), cyclin-D1 (#2978, Cell Signaling Technology), ubiquitin (#3936, Cell Signaling Technology), GFP (#2956, Cell Signaling Technology), CDK2 (sc-6248, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CDK4 (sc-260, Santa Cruz Biotechnology), CDK6 (sc-7961, Santa Cruz Biotechnology), phosphorylated-c-Myc T58 (AF3055, Affinity, Cincinnati, OH, USA), HA (51064-2-AP, Proteintech, Wuhan, China), and Flag (M20008, Abmart, Shanghai, China).
Flag tag (flag)
Manufactured by Abmart
Sourced in China
Flag is a lab equipment designed to hold and display samples or materials during laboratory experiments or analysis. It provides a stable platform to mount and position specimens for observation, measurement, or further processing.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Ubiquitin, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Mical l2, by Thermo Fisher Scientific (1 mentions)
Ha 51064 2 ap, by Proteintech (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
α tubulin, by Merck Group (1 mentions)
Bmpr2, by Abcam (1 mentions)
Smad4, by Proteintech (1 mentions)
Phospho smad1 5, by Cell Signaling Technology (1 mentions)
β actin, by Huabio (1 mentions)
Total smad5, by Cell Signaling Technology (1 mentions)
Total smad2 3, by Cell Signaling Technology (1 mentions)
Phospho smad2, by Cell Signaling Technology (1 mentions)
Sb431542, by Selleck Chemicals (1 mentions)
Ldn193189, by Selleck Chemicals (1 mentions)
6 protocols using flag tag (flag)
1
Western Blot Analysis of Protein Expression
2
Western Blotting of Stem Cell Markers
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Western blotting was carried out with following primary antibodies: Cited1 (made in our lab or Genetex, GTX114559), Oct4 (made in our lab), Sox2 (made in our lab), phospho-Smad1/5 (Cell Signaling Technology, #9516), phospho-Smad2 (Cell Signaling Technology, #3101), total Smad5 (Cell Signaling Technology, #9517), total Smad2/3 (Cell Signaling Technology, #3102), Smad4 (Proteintech, 10231–1-AP), Bmpr2 (Abcam, ab96826), Elf5 (Santa Cruz, sc-9645), T (R&D, AF2085), Placental lactogen 1 (Santa Cruz, sc-376436), Flag (Abmart, M20018F), β-Actin (HuaBio, M1210–2) and α-Tubulin (Sigma, T5168). The inhibitors were purchased from STEMCELL (SB431542: #72232), Selleckchem (LDN193189: S2618; K02288: S7359; protease inhibitor cocktail: B14002; phosphatases inhibitor: B15002) and PeproTech (Noggin: #120–10 C), respectively.
3
Immunofluorescence Staining Protocol
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Cells were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, and blocked with 10% horse serum. Antibodies to hemagglutinin (Cell Signaling Technology, Danvers, MA or Abmart, Shanghai, China) and flag (Abmart) were used as primary antibodies. Alexa Fluor 488–conjugated donkey antimouse IgG (Thermo Fisher Scientific, Eugene, OR) and Alexa Fluor 546‒conjugated goat anti-Rabbit IgG (Thermo Fisher Scientific) were used as secondary antibodies. DAPI (Beyotime Biotech, Suzhou, China) was used as a counterstain to label cell nuclei. Fluorescence was detected using Nikon A1.
4
Comprehensive Antibody-Based Western Blot
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Western blot was performed as previously described [19 ]. Specific primary antibodies against PRMT1 (11279-1-AP, Proteintech), PGK1 (17811-1-AP, Proteintech), GAPDH (60004-1-AP, Proteintech), ADMA ( 13522 S, Cell Signaling Technology), Flag (M29998, Abmart), pS203-PGK1 (SAB487P, Signalway Antibody), meR206-PGK1 (SAB487P-1, Signalway Antibody), PDHA1 (A22081, Abclonal); p-PDHA1 (AP1022, Abclonal); PDHK1 (A0834, Abclonal); p-PDHK1 (11596, Signalway Antibody) ERK1/2 (4695 S, Cell Signaling Technology, short for ERK), Phospho- ERK1/2 (Thr202/Tyr204) (4370 S, Cell Signaling Technology, short for p-ERK) were used for western blot assays. ERK specific inhibitor SCH772984 and PRMT1 inhibitor GSK3368715 were purchased from MedChemExpress (HY-50846, HY-128717A, MCE).
The anti-ADMA-R206-PGK1 (anti-meR206-PGK1) antibody was raised against the region near R206 asymmetric dimethylarginine site of PGK1. The asymmetric di-methylated synthetic peptide [C-NYFAKALESPE(R-Me2,asym)PFLA] was used for immunization in the mice. The antibody was generated in Gl biochem company (Shanghai, China).
The anti-ADMA-R206-PGK1 (anti-meR206-PGK1) antibody was raised against the region near R206 asymmetric dimethylarginine site of PGK1. The asymmetric di-methylated synthetic peptide [C-NYFAKALESPE(R-Me2,asym)PFLA] was used for immunization in the mice. The antibody was generated in Gl biochem company (Shanghai, China).
5
Immunoblotting Antibody and Protein Sourcing
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The His and GST antibodies were purchased from Abmart (Beijing, China). The GAPDH antibody was purchased from Sanjian (Tianjin, China). The FLAG and RGS-His antibodies were purchased from Abmart and Qiagen, respectively. The E1, FLAG-Ub, WT Ub, Lys48-linked Ub2, Ub3, Ub4 and Ub2-7 proteins were purchased from Boston Biochem (Cambridge, MA, USA).
6
Alisertib Modulates Aurora A and Jak/STAT Signaling
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The effect of alisertib on Aurora A and the Jak/STATs signaling pathways was assessed by immunoblotting, as described previously 21 (link), 23 , using the following phospho-specific antibodies: p-Aurora A/B/C (Cell Signaling Technology, #2914), Aurora A (35C1) (Abcam, ab13824), p-Jak1 (T1022/1023) (Cell Signaling Technology, #3331), Jak1 (6G4) (Cell Signaling Technology, #3344), p-Jak2 (T1007/1008) (Cell Signaling Technology, #3776), Jak2 (D2E12) (Cell Signaling Technology, #3230), p-STAT1 (T701) (Cell Signaling Technology, #7649), STAT1 (D1K9Y) (Cell Signaling Technology, #14994), p-STAT2 (T690) (Cell Signaling Technology, #4441), STAT2 (D9J7L) (Cell Signaling Technology, #72604), PARP (46D11) (Cell Signaling Technology, #9532), DNMT3B (D7070) (Cell Signaling Technology, #67259), DNMT1 (Proteintech., 24206-1-AP), DNMT3A (Proteintech., 19366-1-AP), ubiquitin (Proteintech., 10201-1-AP), UHRF1(Proteintech., 21402-1-AP), flag (Abmart, M20008L) and β-actin (Proteintech.,66009-1-Ig).
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