Protein was extracted by RIPA buffer (Beyotime), and separated using 10% SDS-PAGE gel. After electrophoretically transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA), the membranes were incubated with the primary antibodies against proliferating cell nuclear antigen (PCNA; 1:3,000, Genetex, SA, Texas, USA), CyclinD1 (1:2,000, Genetex), B-cell lymphoma-2 (Bcl-2; 1:3,000, Genetex), cleaved-caspase 3 (c-caspase 3, 1:1,000, Genetex), metal matrix proteinase 2 (MMP2; 1:3,000, Genetex), MMP9 (1:2,000, Genetex), HOXC8 (1:3,000, Genetex) or GAPDH (1:5,000, Genetex), followed by incubation with secondary antibody (1:2,000, Genetex). Finally, protein signals were detected using the Supersignal West Femto Kit (Thermo Fisher Scientific).
Cyclin d1
Manufactured by GeneTex
Sourced in United States, Denmark
Cyclin D1 is a protein that plays a crucial role in the cell cycle. It functions as a regulatory subunit of cyclin-dependent kinases, which are essential for the progression of cells through the G1 phase and the G1/S transition of the cell cycle.
Automatically generated - may contain errors
Lab products found in correlation
Proliferating cell nuclear antigen (pcna), by GeneTex (1 mentions)
Supersignal west femto kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Gapdh, by GeneTex (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Beta actin, by GeneTex (1 mentions)
Erk1 2, by GeneTex (1 mentions)
Foxo3, by GeneTex (1 mentions)
Cyclin a2, by GeneTex (1 mentions)
Cyclin b1, by GeneTex (1 mentions)
Bca protein assay kit, by CoWin Biotech (1 mentions)
Tubulin, by GeneTex (1 mentions)
Anti mouse igg, by GeneTex (1 mentions)
Active caspase3, by Proteintech (1 mentions)
Anti rabbit igg, by GeneTex (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
P akt, by Proteintech (1 mentions)
4 protocols using cyclin d1
1
Western Blot Analysis of Protein Markers
2
Lung Cancer Cell Culture and Antibody Characterization
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Lung adenocarcinoma cells HCC827, H1975, H1650, and A549 and nonmalignant lung cells 16HBE were purchased from Nanjing Cobioer Co., Ltd. HCC827, H1975, and H1650 were cultured in RPMI-1640 medium enriched with 10% FBS and allowed to grow under 37°C and 5% CO2 conditions. However, A549 and 16HBE were cultured in F12K and DMEM/high glucose medium enriched with 10% FBS under 37°C and 5% CO2 conditions. We purchased the antibodies of NMU (Affinity, Cat No. DF4238), p-Erk1/2 (Cell signaling technology (CST), Cat No. 4370), Erk1/2 (GeneTex, Cat No. GTX59618), p-FoxO3 (CST, Cat No. 13129), FoxO3 (CST, Cat No. 2493), cyclin A2 (CST, Cat No. 4656), cyclin B1 (CST, Cat No. 12231), cyclin D1 (CST, Cat No. 2922), P21 (CST, Cat No. 2947), P27 (CST, Cat No. 3686), and beta-actin (CST, Cat No. 58169).
3
Immunohistochemical Analysis of CREPT, Cyclin D1, and TCF4
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The EnVision two step immunohistochemical kit was obtained from Dako (Glostrup, Denmark); CREPT, cyclin D1, and TCF4 antibodies were obtained from GeneTex (TX, USA); and the total RNA extraction, reverse transcription, and BCA protein assay kits were obtained from Beijing ComWin Biotech Co. Ltd. (Beijing, China). The 7900 real-time quantitative polymerase chain reaction (RT-PCR) system was obtained from ABI (Foster City, CA, USA) and the RM2235 slicing instrument was obtained from Leica (Solms, Germany).
4
Protein Expression Analysis in Transfected Cells
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Following transfection for 48 h, cell lysates were prepared using RIPA lysis buffer and protease cocktail inhibitor I (Merck KGaA). The protein was separated using 10% SDS-PAGE and subsequently transferred to a PVDF membrane. After blocking in a 5% non-fat milk for 1 h, the membrane was washed with TBS + 0.1% Tween-20. Membranes were incubated with primary antibodies against METTL3 (1:1,000; cat. no. GTX105037; GeneTex, Inc.), Bcl-2 (1:2,000; cat. no. 60178-1-Ig; ProteinTech Group, Inc.); Bax (1:1,000; cat. no. 50599-2-Ig; ProteinTech Group, Inc.); active caspase3 (1:1,000; rabbit polyclonal antibody; cat. no. 19677-1-AP; ProteinTech Group, Inc.); p-AKT (1:1,000; cat. no. 66444-1-Ig; ProteinTech Group, Inc.); AKT (1:500; cat. no. 9272; Cell Signaling Technology, Inc.); p70S6K (1:1,000; cat. no. GTX107562; GeneTex, Inc.); Cyclin D1 (1:1,000; cat. no. GTX108624; GeneTex, Inc.) and tubulin (1:1,000; cat. no. GTX76511; GeneTex, Inc.) at 4°C overnight, followed by incubation with anti-rabbit IgG (1:2,000; cat. no. GTX300119; GeneTex, Inc.) or anti-mouse IgG (1:2,000; cat. no. GTX300120; GeneTex, Inc.) secondary antibodies for 1 h at room temperature. Protein bands were visualized using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.). Protein band intensity was analyzed using Image J software, v1.41 (National Institutes of Health).
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