Cyclin d1

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom, Germany, China, Canada, Japan

Cyclin D1 is a core cell cycle regulatory protein that plays a crucial role in the progression of cells from the G1 phase to the S phase of the cell cycle. It functions as a regulatory subunit of the CDK4 and CDK6 kinases, which are essential for the control of the cell cycle.

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Anti-cyclin D1 (HD-11) Mouse anti-cyclin D1 Anti-cyclin D1 M20 Cyclin D1 siRNA Cyclin D1 antibody Mouse antibody against Cyclin D1 Mouse monoclonal anti-cyclin D1 Mouse anti-cyclin D1 clone A-12 Anti-Cyclin D1 monoclonal antibody Antibodies against cyclin D1

512 protocols using cyclin d1

Antibody Generation and Validation for Acetylation-Specific HMGN2

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Rabbit polyclonal ac-K2-HMGN2 antibody was made by New England Peptides. The antibody was raised against an acetylated N-terminal HMGN2 peptide (P(KAc)RKAEGDAC), followed by affinity purification. To ensure specificity, a blocking peptide (P(KAc)RKAEGDAC) and a non-acetyl blocking peptide (PKRKAEGDAC) were used to ensure acetyl-specific interaction. Antibodies used for western blotting; Tubulin (Invitrogen, 322500, 1:2000); HDAC6 (Abcam, ab47181, 1:500); HMGN2 (Millipore, 07-252, 1:500; Cell Signaling, #9437, 1:2000) pan-acetyl lysine (Millipore, AB3879, 1:500); CISH (Santa Cruz, sc-15344, 1:500); CyclinD1 (Santa Cruz, sc-718, 1:500); CEBPβ (Santa Cruz, sc-150, 1:500); PARP/Cleaved PARP (Cell Signaling, #9542, 1:500); ac-K2-HMGN2 (1:50); acetyl-α-Tubulin (Cell Signaling, #5335, 1:500). Secondary antibody was used at a dilution of 1:1000 for all antibodies except Tubulin, which was 1:2000. Antibodies used for IHC: CyclinD1 (Santa Cruz, sc-718, 1:1500); acetyl-Tubulin (Cell Signaling, D20G3, 1:100); ac-K2-HMGN2 (1:500).

Medler T.R., Craig J.M., Fiorillo A.A., Feeney Y.B., Harrell J.C, & Clevenger C.V. (2016). HDAC6 Deacetylates HMGN2 to Regulate Stat5a Activity and Breast Cancer Growth. Molecular cancer research : MCR, 14(10), 994-1008.

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Antibody-based Analysis of Cell Signaling

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The antibodies used in western blot analysis were cyclin D1 (sc-20044, Santa Cruz), b-actin (sc-47778, Santa Cruz), Akt1 (sc-5298, Santa Cruz), pAkt1 Ser473 (#9018, CST), pAkt1/2/3 Ser473 (sc-7985-R, Santa Cruz), TSC2 (sc-893, Santa Cruz), pTSC2 Ser939 (ab59269, Abcam), FKHR (sc-11350, Santa Cruz), pFKHR Ser319 (sc-101682, Santa Cruz), cyclin A2 (ab16726, Abcam), Bad (sc-8044, Santa Cruz), pBad Ser136 (ab28824, Abcam), Rictor (SC#271081, H11) and phosphorylated RB (S780) (Cell Signaling). Mouse anti-FLAG (M2), mouse anti-vinculin (hVIN-1) antibodies were from Sigma (St. Louis, MO). The antibody used for Immunoprecipitation was HA (sc-805, Santa Cruz). The antibodies used for immunohistochemistry and immunofluorescence were cyclin D1 (sc-20044, Santa Cruz), for Akt Ser473 phosphorylation (pAkt1/2/3 Ser473) (sc-7985-R, Santa Cruz), for pAkt1 Ser473 (#9018, CST), TSC2 (sc-893, Santa Cruz), pTSC2 Ser939 (ab59269, Abcam), pFKHR Ser319 (sc-101682, Santa Cruz), pBad Ser136 (ab28824, Abcam), cyclin A2 (ab16726, Abcam), paxillin (05–417, Millipore-Sigma), Rictor (H278, sc-99004), tyrosine phosphorylated paxillin (44–722G, Thermo Fisher), PACSIN2 (10518–2-AP, Proteintech).

Chen K., Jiao X., Di Rocco A., Shen D., Xu S., Ertel A., Yu Z., Di Sante G., Wang M., Li Z., Pestell T.G., Casimiro M.C., Skordalakes E., Achilefu S, & Pestell R.G. (2020). Endogenous Cyclin D1 Promotes the Rate of Onset and Magnitude of Mitogenic Signaling via Akt1 Ser473 Phosphorylation. Cell reports, 32(11), 108151.

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Antibody-based Analysis of Cell Signaling

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Molecular Mechanisms of Drug Interactions

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Methanol, crystal violet, and chloroquine (CQ) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rabbit polyclonal antibodies against cyclin-dependent kinase 4 (CDK4), poly(ADP-ribose) polymerase (PARP), Bcl-2, Bid, Thr(P)172-adenosine monophosphate-activated protein kinase (AMPK), AMPK, Ser(P)2448-mammalian target of rapamycin (mTOR), mTOR, Ser(P)79-acetyl-CoA carboxylase (ACC), ACC, fatty acid synthase (FAS), microtubule-associated light chain 3 (LC3), and p62 were obtained from Cell Signaling (Beverly, MA, USA); p21 and cyclin D1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Ser(P)872-hydroxymethylglutaryl-CoA reductase (HMGCR), and HMGCR were from Millipore (Billerica, MA, USA); cyclin D1 was from Santa Cruz Biotechnology; and ANGPTL4 was from Abcam (Cambridge, MA, USA). Mouse monoclonal antibodies against beta-actin and caspase-3 were, respectively, purchased from MP Biomedicals (Irvine, CA, USA) and Imgenex (San Diego, CA, USA).

Tsai W.L., Wang C.Y., Lee Y.C., Tang W.C., Anuraga G., Ta H.D., Wu Y.F, & Lee K.H. (2021). A New Light on Potential Therapeutic Targets for Colorectal Cancer Treatment. Biomedicines, 9(10), 1438.

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Immunohistochemical Analysis of Cholesteatoma

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The tissue samples were sliced at a thickness of 4 μm. The sections were incubated with primary antibody (STAT3, surviving, VEGF (all from Proteintech Group, USA) and cyclin D1 (Santa Cruz Biotechnology, USA)) overnight, followed by a biotin-labelled secondary antibody. The evaluation of immunostaining included both positivity rate and staining intensity of cells [22 (link)]. The proportion of positive cells was graded as follows: 0 for no staining, 1 for < 30%, 2 for 30–60%, and 3 for > 60%. The grading of staining density was 0 for no staining, 1 for light brown, 2 for brown, and 3 for dark brown. All specimen sections were analysed independently by two researchers with the double-blind method. The final score was obtained from the product of the percentage score and the staining intensity score. The final result was evaluated as positive when the score was ≥ 3 or negative when the score was < 3. The cholesteatoma tissues, control skin, and HaCaT cells were lysed with RIPA lysis buffer. Then, the proteins were separated and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA), which was incubated with primary antibody (STAT3, surviving, VEGF, β-actin (all from Proteintech Group, USA) and cyclin D1 (Santa Cruz Biotechnology, USA)). The enhanced chemiluminescence ECL detection system (Thermo Fisher Scientific, MA) was used to present the results.

Zang J., Yang B., Feng S, & Jiang X. (2019). Low expression of microRNA-125b enhances the expression of STAT3 and contributes to cholesteatoma growth. Archives of Medical Science : AMS, 18(6), 1596-1606.

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Deferasirox modulates iron homeostasis

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Deferasirox (Exjade®) was donated by Novartis (Basel, Switzerland). Goat polyclonal anti-NDRG1 (N-myc downstream regulated gene 1) (catalog no. ab37897) and rabbit polyclonal anti-ferroportin (catalog no. ab85370) antibodies were purchased from Abcam (Cambridge, UK). Anti-TFR1 mouse monoclonal antibodies (catalog no. 136800) were obtained from Life Technologies (Carlsbad, CA, USA), and FeSO₄ was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-p53, anti-p27, p21, cyclin A, cyclin B, cyclin D1, cyclin E, CDK2, CDK4, CDK6, c-myc, pro-caspase 3, and BAX antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-mTOR and pro-caspase 8 antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA).

Choi J.H., Kim J.S., Won Y.W., Uhm J., Park B.B, & Lee Y.Y. (2016). The potential of deferasirox as a novel therapeutic modality in gastric cancer. World Journal of Surgical Oncology, 14, 77.

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Comprehensive Immunoblotting Analysis of Muscle Proteins

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Total protein of the cell or tissue samples was harvested using radioimmunoprecipitation lysis buffer after washing three times with PBS. Immunoblotting was carried out according to our previous method (10 (link)). The primary antibodies targeted the following proteins: MyHC (1:500; no. MAB4470; R and D Systems), MyoD (1:500; no. NB100-56511; Novus Biologicals), AKT (1:1000; no. 9272S; CST), p-AKT (Ser473) (1:2000; no. 4060S; CST), PI3K (p55α) (1:1000; no. 11889S; CST), p-PI3K p55 (Tyr199) (1:1000; no. 4228S; CST), β-tubulin (1:200; no. sc-58880; Santa Cruz Biotechnology), MyoG (1:1000; no. NB100-56510SS; Novus Biologicals), MuRF1 (1:200; no. sc-398608; Santa Cruz Biotechnology), MAFbx (1:200; no. sc-166806, Santa Cruz Biotechnology), cyclin E (1:100; no.sc-377100; Santa Cruz Biotechnology), cyclin D1 (1:100; no. sc-450; Santa Cruz Biotechnology), PTEN (1:500; no. sc-7974; Santa Cruz Biotechnology), IGF2BP2 (1:100; no. sc-377014; Santa Cruz Biotechnology), and proliferating cell nuclear antigen (1:100; no. sc-56; Santa Cruz Biotechnology). The secondary antibodies included goat anti-rabbit IgG (1:3000; no. BA1054; BosterBio) and goat antimouse IgG (1:3000; no. BA1050; BosterBio).

Cai R., Zhang Q., Wang Y., Yong W., Zhao R, & Pang W. (2021). Lnc-ORA interacts with microRNA-532-3p and IGF2BP2 to inhibit skeletal muscle myogenesis. The Journal of Biological Chemistry, 296, 100376.

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Western Blot Analysis of Protein Expression

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Total protein was extracted and quanti ed. Twenty-ve micrograms of protein was separated by 10% g polyacrylamide gel electrophoresis (PAGE), and then transferred to a nitrocellulose membrane. Skim milk 5% was used to block non-speci c antigen binding, and then primary antibodies were added (RASSF1A, Abcam, ab23950; YAP, Cell Signaling Technology, #4912; pS127-YAP, Cell Signaling Technology, #13008; Cyclin A, Santa Cruz, sc-596; Cyclin B1, Proteintech, 55004-1-AP; Cyclin D1, Santa Cruz, sc-246; Cyclin E, Santa Cruz, sc-25303; CDK1, Wanleibio, WL02373; cleaved-caspase-3, Wanleibio, WL02348; Bcl-2, Wanleibio, WL01556; BAX, Proteintech, 50599-2-lg; p73, Wanleibio, WL01604; p53, Santa Cruz, sc-126; p21, Wanleibio, WL0362; AKT, Wanleibio, WL0003b; p-AKT, Wanleibio, WLP001a; ERK, Wanleibio, WL01864; p-ERK, Wanleibio, WLP1512; STAT3, Wanleibio, WL03207; p-STAT3, Wanleibio, WLP2412; NF-κB P65, Wanleibio, WL01980; β-actin, Santa Cruz, sc-47778; Histone H3, Wanleibio) and incubated at 4 °C overnight. Next, secondary antibodies were added and incubated at room temperature for 1 h, and nally exposed on X-ray lm.

Jia J., Wang N., Zheng Y., Mo X., Zhang Y., Ye S., Liu J., Yan F., Li H., & Chen D. (2020). RASSF1A regulates the abnormal cell proliferation in psoriasis via inhibition of YAP.

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Characterizing Cell Signaling Pathways

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The following reagents used in this study were purchased from the indicated sources: cycloheximide (Sigma, 20 μg/ml), doxorubicin (Sigma, 0.5 μg/ml), doxycycline (Sigma, 1 μg/ml), Hoechst 33342 (Sigma, 1 μg/ml), lipofectamine 2000 (Invitrogen), streptavidin-coated agarose beads (Thermo Fisher Scientific), complete EDTA-free protease inhibitor cocktail (Roche Applied Science), antibodies against GAPDH (Santa Cruz, sc-166545, 1:5000), p53 (Santa Cruz, sc-126, 1:1000), p21 (Sigma, #P1484, 1:2000), Flag (Sigma, #F3165, 1:4000), p27 (Abcam, Y236, 1:1000), cyclin D1 (Santa Cruz, sc-753, 1:1000), cyclin E1 (Santa Cruz, sc-481, 1:1000), PTBP1 (Proteintech, 12582-1-AP, 1:1000), HRP-conjugated secondary antibodies against mouse (115-035-062), and rabbit (111-035-144) (Jackson ImmunoResearch, 1:10000).

Yang Y., Wang C., Zhao K., Zhang G., Wang D, & Mei Y. (2018). TRMP, a p53-inducible long noncoding RNA, regulates G1/S cell cycle progression by modulating IRES-dependent p27 translation. Cell Death & Disease, 9(9), 886.

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Western Blot Analysis of Activated HSCs

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As described previously with modifications [28 ], the aHSCs were lysed in a lysis buffer (Sigma-Aldrich, Missouri, USA). The lysed cells were centrifuged at 15,000 rpm for 15 min at 4°C; then, the supernatants (30 μg protein) were loaded to SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked using 1% BSA in PBS containing 0.1% Tween-20 for 1 h at room temperature. The proteins were detected through an overnight incubation at 4°C with primary antibodies against a-SMA (mouse monoclonal), β-actin (mouse monoclonal) (Sigma-Aldrich, Missouri, USA), collagen I (mouse monoclonal), cyclin D-1 (rabbit polyclonal), p27 (rabbit polyclonal), SREBP1 (mouse monoclonal) (Santa Cruz Biotechnology, CA), and PPARγ (rabbit monoclonal) (Cell Signaling Technology, Beverly, MA). Finally, the membrane was incubated with a HRP-conjugated secondary antibody for 1 h. An enhanced chemiluminescence kit (Millipore, Bedford, MA) was used to visualize the reactive signals, quantified by ImageJ software.

Lee S.L., Hsu W.H., Tu C.M., Wang W.H., Yang C.Y., Lee H.K, & Chin T.Y. (2021). The Effects of Freshwater Clam (Corbicula fluminea) Extract on Activated Hepatic Stellate Cells. Evidence-based Complementary and Alternative Medicine : eCAM, 2021, 6065168.

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