rabbit anti cdc37  (Proteintech)

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rg5 enhances Abemaciclib sensitivity in ER+ breast cancer by modulating cell cycle proteins via transcriptional and post-translational levels

doi: 10.1016/j.jgr.2025.06.004

Figure Lengend Snippet: Ginsenoside Rg5 interrupts the binding between HSP90 and CDC37 to facilitate the degradation of cell cycle related proteins A. HSP90 and CDC37 protein levels in MCF-7, MCF-7/AR, T47D, and T47D/AR cells treated with DMSO or Rg5 (40 μM) for 24 h. B. Co-IP was performed using an anti-HSP90 antibody to pull down HSP90 protein complexes from MCF-7/AR and T47D/AR cell lysates treated with DMSO or Rg5 (10 or 40 μM) for 24 h. Western blotting was then used to detect the presence of CDC37 in the immunoprecipitated complexes. C. Cellular Thermal Shift Assay (CETSA) was used to determine the thermal stability of HSP90 in MCF-7/AR and T47D/AR cells treated with DMSO or Rg5 (40 μM) for 24 h. Cells after treatment were heated at the indicated temperatures (45 °C, 49 °C, 53 °C, 56 °C, 59 °C, 62 °C, and 65 °C), followed by immunoblot analysis to detect HSP90 and β-actin levels. D-I. MCF-7/AR (D–F) and T47D (G–I) cells treated with DMSO, 17-AAG (10 μM), or Rg5 (40 μM) for 24 h were subjected to cycloheximide (CHX, 50 μg/mL) chase analysis to monitor the stability of CDK4 and CDK6 proteins over time (up to 24 h). D-G. Western blot analyses of CDK4 and CDK6 protein levels in MCF-7/AR (D) and T47D/AR (G) cells after CHX treatment. E-F and H-I. Quantitative analysis of CDK4 and CDK6 protein levels in MCF-7/AR (E–F) and T47D/AR (H–I) cells over the CHX chase time course. Data are presented as mean ± SD from three independent experiments. ∗comparison between 17-AAG and DMSO groups; # comparison between ginsenoside Rg5 and DMSO groups. ## p < 0.01, ∗∗∗ and ### p < 0.001.

Article Snippet: These membranes were incubated with primary antibodies targeting cleaved caspase-3 (1:1000, #9661, CST), cleaved caspase-9 (1:1000, #9509, CST), p-AKT (Ser473, 1: 1000, #4060, CST), AKT (1:2000, 10176-2-AP, Proteintech), p-mTOR (Ser2448, 1:1000, #5536, CST), mTOR (1:1000, #2983, CST), CDK2 (1:5000, 10122-1-AP, Proteintech), CDK4 (1:2000, 11026-1-AP, Proteintech), CDK6 (1: 1000, 14052-1-AP, Proteintech), cyclin E1 (1:1000, 11554-1-AP, Proteintech), cyclin D1 (1:5000, 60186-1-Ig, Proteintech), HSP90 (1:2000, 13171-1-AP, Proteintech), CDC37 (1:1000, #4793, CST), β-actin (1:5000, 20536-1-AP, Proteintech) and GAPDH (1:5000, 10494-1-AP, Proteintech).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation, Thermal Shift Assay, Comparison

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rg5 enhances Abemaciclib sensitivity in ER+ breast cancer by modulating cell cycle proteins via transcriptional and post-translational levels

doi: 10.1016/j.jgr.2025.06.004

Figure Lengend Snippet: HSP90A-Y61A mutant canceled the regulative effect of ginsenoside Rg5 on enhancing Abemaciclib sensitivity in vitro A. mRNA levels of HSP90 in MCF-7/AR and T47D/AR cells transfected with shNC (control), shHSP90A, shHSP90A + Vector, or shHSP90A + HSP90A-Y61A constructs. B. Western blot analysis showing protein levels of HSP90 in MCF-7/AR and T47D/AR cells treated with the indicated constructs. C. Co-IP was performed using an anti-HSP90 antibody to pull down HSP90 protein complexes from MCF-7/AR and T47D/AR cells with knock down of endogenous HSP90A and enforced expression of HSP90A mutant Y61A (shHSP90A + HSP90A-Y61A), with or without ginsenoside Rg5 treatment (40 μM) for 24 h. Western blot used to detect the presence of CDC37 in the immunoprecipitated complexes. D. MCF-7/AR and T47D/AR cells with knock down of endogenous HSP90A and enforced expression of HSP90A mutant Y61A (shHSP90A + HSP90A-Y61A) were treated with DMSO, 17-AAG (10 μM), or Rg5 (40 μM) for 24 h were subjected to cycloheximide (CHX, 50 μg/mL) chase analysis to monitor the stability of CDK4 and CDK6 proteins over time (0–24 h). E-H. Quantitative analysis of CDK4 and CDK6 protein levels in MCF-7/AR (E, F) and T47D/AR (G, H) cells covered in panel (D) over the CHX chase time course. I-J. Abemaciclib IC50 in MCF-7/AR (I) and T47D/AR (J) cells with indicated combination of HSP90A knockdown, restoration of HSP90A-Y61A, and treatment with ginsenoside Rg5 (40 μM) for 72 h. K-L. Cell cycle distribution analysis of MCF-7/AR (K) and T47D/AR (L) cells treated with shNC, shHSP90A, shHSP90A + Vector, shHSP90A + HSP90A-Y61A, and shHSP90A + HSP90A-Y61A + Rg5 (40 μM). M-P. Colony formation assays of MCF-7/AR (M, O) and T47D/AR (N, P) cells treated with shNC, shHSP90A, shHSP90A + Vector, shHSP90A + HSP90A-Y61A, and shHSP90A + HSP90A-Y61A + Rg5 (40 μM) in the presence or absence of Abemaciclib (1 μM). ∗∗∗ p < 0.001 for 17-AAG vs. DMSO; ## p < 0.01; ### p < 0.001 for Rg5 vs. DMSO.

Article Snippet: These membranes were incubated with primary antibodies targeting cleaved caspase-3 (1:1000, #9661, CST), cleaved caspase-9 (1:1000, #9509, CST), p-AKT (Ser473, 1: 1000, #4060, CST), AKT (1:2000, 10176-2-AP, Proteintech), p-mTOR (Ser2448, 1:1000, #5536, CST), mTOR (1:1000, #2983, CST), CDK2 (1:5000, 10122-1-AP, Proteintech), CDK4 (1:2000, 11026-1-AP, Proteintech), CDK6 (1: 1000, 14052-1-AP, Proteintech), cyclin E1 (1:1000, 11554-1-AP, Proteintech), cyclin D1 (1:5000, 60186-1-Ig, Proteintech), HSP90 (1:2000, 13171-1-AP, Proteintech), CDC37 (1:1000, #4793, CST), β-actin (1:5000, 20536-1-AP, Proteintech) and GAPDH (1:5000, 10494-1-AP, Proteintech).

Techniques: Mutagenesis, In Vitro, Transfection, Control, Plasmid Preparation, Construct, Western Blot, Co-Immunoprecipitation Assay, Knockdown, Expressing, Immunoprecipitation

Journal: Journal of Ginseng Research

Article Title: Ginsenoside Rg5 enhances Abemaciclib sensitivity in ER+ breast cancer by modulating cell cycle proteins via transcriptional and post-translational levels

doi: 10.1016/j.jgr.2025.06.004

Figure Lengend Snippet: HSP90A-Y61A mutant canceled the regulative effect of ginsenoside Rg5 on enhancing Abemaciclib sensitivity in vivo A. Representative images of T47D/AR xenograft tumors harvested from mice after treatment with different combinations: shNC (control), shNC + Abemaciclib (Abe, 20 mg/kg/day), shHSP90A, shHSP90A + HSP90A-Y61A, shNC + Abe + Rg5 (20 mg/kg/day), shHSP90A + Y61A + Abe, and shHSP90A + Y61A + Abe + Rg5. B. Tumor weight measurements of T47D/AR derived tumors from different treatment groups on day 36. Data are shown as mean ± SD (n = 5 or 6). C. Tumor volume growth curves of T47D/AR xenografts from different treatment groups over time (mean ± SD, n = 5 or 6). D. Immunohistochemistry (IHC) analysis of xenograft tumor sections from the indicated treatment groups, showing staining for Ki-67, CDK4, CDK6, and CDC37. Hematoxylin and eosin (H&E) staining is also shown. Scale bars represent 50 μm. E. Western blot analysis of HSP90, CDK4, and CDK6 protein expression in indicated tumor samples from each treatment group. ∗∗∗ p < 0.001.

Article Snippet: These membranes were incubated with primary antibodies targeting cleaved caspase-3 (1:1000, #9661, CST), cleaved caspase-9 (1:1000, #9509, CST), p-AKT (Ser473, 1: 1000, #4060, CST), AKT (1:2000, 10176-2-AP, Proteintech), p-mTOR (Ser2448, 1:1000, #5536, CST), mTOR (1:1000, #2983, CST), CDK2 (1:5000, 10122-1-AP, Proteintech), CDK4 (1:2000, 11026-1-AP, Proteintech), CDK6 (1: 1000, 14052-1-AP, Proteintech), cyclin E1 (1:1000, 11554-1-AP, Proteintech), cyclin D1 (1:5000, 60186-1-Ig, Proteintech), HSP90 (1:2000, 13171-1-AP, Proteintech), CDC37 (1:1000, #4793, CST), β-actin (1:5000, 20536-1-AP, Proteintech) and GAPDH (1:5000, 10494-1-AP, Proteintech).

Techniques: Mutagenesis, In Vivo, Control, Derivative Assay, Immunohistochemistry, Staining, Western Blot, Expressing