Dulbecco’s Modified Eagle medium (DMEM)/F-12 1:1 Modified medium (DMEM/F-12) for the cell culture was purchased from Lonza (Walkersville, MD, USA). LiCl, MG132, PD98059, SB230580, SP600125, LY294002, BAY 11–7280, leptomycin B (LMB) and 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and N-acetyl-L-cysteine (NAC) were purchased from Sigma Aldrich (St. Louis, MO, USA). Antibodies against cyclin D1, phospho-cyclin D1 (Thr286), HA-tag, CRM1 and β-actin were purchased from Cell Signaling (Bervely, MA, USA). All chemicals were purchased from Fisher Scientific, unless otherwise specified.
Antibodies against cyclin d1
Manufactured by Cell Signaling Technology
Sourced in United States
Antibodies against cyclin D1 are laboratory reagents designed to detect and study the cyclin D1 protein, which is a key regulator of the cell cycle. These antibodies can be used in various research applications, such as Western blotting, immunohistochemistry, and flow cytometry, to analyze the expression and localization of cyclin D1 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
P yap, by Cell Signaling Technology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Antibody for fth, by Abcam (2 mentions)
Antibody for n myc down stream regulated gene 1 ndrg1, by Proteintech (2 mentions)
Propidium iodide buffer, by Thermo Fisher Scientific (2 mentions)
C myc, by Cell Signaling Technology (2 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (2 mentions)
P mek1 2, by Cell Signaling Technology (2 mentions)
P c raf, by Cell Signaling Technology (2 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Lithium chloride (licl), by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Sp600125, by Merck Group (1 mentions)
Ly294002, by Merck Group (1 mentions)
Pd98059, by Merck Group (1 mentions)
Dulbecco s modified eagle medium dmem f 12 1 1 modified medium dmem f 12, by Lonza (1 mentions)
Leptomycin b lmb, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
6 protocols using antibodies against cyclin d1
1
Investigating Cyclin D1 Regulation
2
Protein Expression Quantification by Western Blot
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Antibodies against P53 (#10442) were purchased from Protein Tech (Chicago, IL, USA). Antibodies against P57 (#DF2993), and P21 (#AF6290) were purchased from Affinity Bioscience (USA). Antibodies against GCNT4 (#138788) were purchased from Sigma-Aldrich (Darmstadt, Germany). Antibodies against cyclin D1 (#2978), cyclin B1 (#4138), cdc25B (#9525), and GAPDH (#2118) were bought from Cell Signalling Technology (Cambridge, MA, USA).
Cells were lysed in RIPA buffer (Sigma-Aldrich, Darmstadt, Germany), which contain a phosphatase inhibitor (Roche, Basel, Switzerland) and a protease inhibitor (Roche, Basel, Switzerland). As mentioned above, the BCA Protein Assay Kit (Thermo Scientific, USA) was selected to measure protein concentration.19 (link) The cell lysates were added with sample buffer and boiled at 95°C for 5 min. The samples were transferred to 12% SDS-PAGE at 80 V for 3 h and then electrophoretically transferred to PVDF membranes (Millipore, Billerica, MA, USA) for another 2 h. The membranes then were blocked by 5% BSA for 30 min and incubated with primary antibodies at 4°C overnight and washed by 1% TBST for three times followed by incubated with secondary antibodies (1:2000, Cell Signaling Technology) for 1 h. The final detection of the substrates was performed with the ECL system (#32209, Thermo Fisher, USA). Protein expression levels were normalized to that of GAPDH.
Cells were lysed in RIPA buffer (Sigma-Aldrich, Darmstadt, Germany), which contain a phosphatase inhibitor (Roche, Basel, Switzerland) and a protease inhibitor (Roche, Basel, Switzerland). As mentioned above, the BCA Protein Assay Kit (Thermo Scientific, USA) was selected to measure protein concentration.19 (link) The cell lysates were added with sample buffer and boiled at 95°C for 5 min. The samples were transferred to 12% SDS-PAGE at 80 V for 3 h and then electrophoretically transferred to PVDF membranes (Millipore, Billerica, MA, USA) for another 2 h. The membranes then were blocked by 5% BSA for 30 min and incubated with primary antibodies at 4°C overnight and washed by 1% TBST for three times followed by incubated with secondary antibodies (1:2000, Cell Signaling Technology) for 1 h. The final detection of the substrates was performed with the ECL system (#32209, Thermo Fisher, USA). Protein expression levels were normalized to that of GAPDH.
3
Protein Extraction and Western Blotting Analysis
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Total proteins from cell lines or fresh lung cancer/normal tissues were extracted in lysis buffer and quantified using the Bradford method. 30 mg protein was separated by SDS-PAGE. Nuclear/ cytoplasmic protein were separated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo scientific, USA) according to the manufacturer's protocol. Samples were transferred to PVDF membranes (Millipore, Billerica, MA, USA) and incubated overnight at 4°C with primary antibodies. Antibodies against cyclin D1, cyclin E, p27, CDK4, CDK6, CTGF, p-YAP, YAP (1:1000 dilution) and GAPDH (1:2000 dilution) were obtained from Cell Signaling Technology (Beverly, MA, USA). Rab11a antibody was from Proteintech (Proteintech, USA). After incubation with HRP-coupled anti-mouse or rabbit IgG antibody at 37°C for 2 hours. Target proteins on PVDF membrane were visualized using Pierce ECL kit and captured using a DNR BioImaging System (DNR, Jerusalem, Israel).
For immunoprecipitation, Magnetic Beads (Bio-Rad SureBeads) were incubated with antibodies and unbound antibodies were washed away. Then beads-antibody complex was incubated with target protein. The beads were magnetized using SureBeads magnetic rack and supernatant was discarded. Then elution buffer was used to collect purified target protein for western blot analysis.
For immunoprecipitation, Magnetic Beads (Bio-Rad SureBeads) were incubated with antibodies and unbound antibodies were washed away. Then beads-antibody complex was incubated with target protein. The beads were magnetized using SureBeads magnetic rack and supernatant was discarded. Then elution buffer was used to collect purified target protein for western blot analysis.
4
Antibody-based Protein Expression Analysis
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Antibodies against Cyclin D1, CDK2, CDC6, pSrc, Src, pp38, p38, pYAP, YAP and AXL were purchased from Cell Signal Technology. Antibodies against CYR61 were purchased from Santa Cruz Biotechnology. Antibodies against Ki67 were purchased from Bioworld Technology. Antibodies against Lamin A+C, GAPDH and α-Tubulin, as well as secondary antibodies, were purchased from Huabio Biotechnology. UM-164 was purchased from Selleck. Cell Counting Kit-8 (CCK8) was purchased from Apexbio Technology. Nuclear and cytoplasmic extraction reagents were purchased from ThermoFisher Scientific.
5
Transferrin Receptor Modulation in Cell Studies
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The antibody for transferrin receptor 1 (TfR1) was purchased from Invitrogen (Carlsbad, CA, USA). Antibody for FTH was purchased from Abcam (Cambridge, MA, USA). Antibodies against cyclin D1, cyclin E, proliferating cell nuclear antigen (PCNA), c-myc, p-ERK, ERK, p-c-Raf, and p-MEK1/2 were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody for N-myc down-stream-regulated-gene 1 (NDRG1) was purchased from Proteintech (Wuhan, China). All secondary antibodies used for western blotting and immunohistochemistry were purchased from KPL (Gaithersburg, MD, USA). Cell counting kit-8 (CCK-8) was purchased from MCE (Princeton, NJ, USA). PE Annexin V Apoptosis Detection Kit I was purchased from BD Biosciences (San Diego, CA, USA). Propidium iodide (PI) buffer was purchased from Invitrogen (Carlsbad, CA, USA). DFX was obtained from Novartis (Basel, Switzerland). For in vitro studies, DFX was dissolved in dimethyl sulfoxide at a stock concentration of 100 mM. For in vivo studies, DFX was dissolved in a sodium chloride solution (0.9% w/v; CSPC PHARMA. Shijiazhuang, Hebei, China).
6
Transferrin Receptor Modulation in Cell Studies
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The antibody for transferrin receptor 1 (TfR1) was purchased from Invitrogen (Carlsbad, CA, USA). Antibody for FTH was purchased from Abcam (Cambridge, MA, USA). Antibodies against cyclin D1, cyclin E, proliferating cell nuclear antigen (PCNA), c-myc, p-ERK, ERK, p-c-Raf, and p-MEK1/2 were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody for N-myc down-stream-regulated-gene 1 (NDRG1) was purchased from Proteintech (Wuhan, China). All secondary antibodies used for western blotting and immunohistochemistry were purchased from KPL (Gaithersburg, MD, USA). Cell counting kit-8 (CCK-8) was purchased from MCE (Princeton, NJ, USA). PE Annexin V Apoptosis Detection Kit I was purchased from BD Biosciences (San Diego, CA, USA). Propidium iodide (PI) buffer was purchased from Invitrogen (Carlsbad, CA, USA). DFX was obtained from Novartis (Basel, Switzerland). For in vitro studies, DFX was dissolved in dimethyl sulfoxide at a stock concentration of 100 mM. For in vivo studies, DFX was dissolved in a sodium chloride solution (0.9% w/v; CSPC PHARMA. Shijiazhuang, Hebei, China).
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