anti hsp90a b  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: Bombinin-BO1 induces hepatocellular carcinoma cell-cycle arrest and apoptosis via the HSP90A-Cdc37-CDK1 axis.

doi: 10.1016/j.isci.2024.110382

Figure Lengend Snippet: Figure 3. HSP90A serves as the target of BO1 (A) SDS-PAGE analysis of pull-down samples. (B) Venn plot. Samples were analyzed by LC-MS/MS mass spectrometry and screened for proteins specific to the bio-BO1 pull-down samples in combination with FDR<0.01 and Score Sequest HT > 80. (C) The abundance of HSP90A in samples was analyzed by western blot. Input: whole protein from cell lysis, NC: sample pulled by IgG, bio-BO1: sample pulled by bio-BO1. (D and E) The localization of FITC-BO1 and HSP90A in Hep 3B and Huh7 cells was detected by confocal microscopy. green: FITC-BO1, red: HSP90A, yellow: co- localization of FITC-BO1 and HSP90A, scale bar: 20 mm.

Article Snippet: Materials purchased from Proteintech Group (Wuhan, China) included antibodies against GAPDH (proteintech, 10494-1-AP, 1:5000), Beta Actin (proteintech, 66009-1-Ig, 1:1000), goat anti-rabbit IgG (proteintech, 66912-1- lg, 1:10000), Pro Caspase-8 (Abcam, ab108333, 1:10000), Caspase-9 (proteintech, 10380-1-AP, 1:1000), Cleaved Caspase-9 (Abcam, ab2324, 1 ug/mL), caspase-3 (proteintech, 19677-1-AP, 1:2000), cleaved caspase-3 (Abcam, ab2302, 1:100), Bcl-2 (proteintech, 12789-1-AP, 1:1000), Bax (proteintech, 50599-2-lg,1:1000), HSP90A (proteintech, 60318-1-Ig, 1:5000), Cdc37 (proteintech, 10218-1-AP, 1:200), CDK1 (proteintech, 19532-1-Ig, 1:200), Cyclin A2 (proteintech, 18202-1-AP, 1:5000).

Techniques: SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Lysis, Confocal Microscopy

Journal: iScience

Article Title: Bombinin-BO1 induces hepatocellular carcinoma cell-cycle arrest and apoptosis via the HSP90A-Cdc37-CDK1 axis.

doi: 10.1016/j.isci.2024.110382

Figure Lengend Snippet: Figure 4. BO1 induces the degradation of CDKs by regulating HSP90A-Cdc37 (A and B) Putative binding site analysis of BO1 on HSP90A_MC or HSP90A_N and the surrounding amino acid residues by molecular docking. (C and D) Whole-cell lysates from BO1-treated cells were immunoprecipitated and analyzed for the interaction of HSP90A, Cdc37, and CDK1 by immunoblotting. (E) The expression of HSP90A in cells was determined by RT-qPCR. (F and G) Cells were pretreated with 20 mM MG-132 for 4 h before treatment with (+) or without () 15 mM BO1 for 24 h, and CDK1 was determined by immunoblotting. (H and I) Overexpression of HSP90A in Hep 3B cells significantly repaired BO1-induced CDK1 reduction. (J and K) Cells were treated with 10 mM NH4Cl for 4 h before treatment with (+) or without () 15 mM BO1 for 24 h, and then CDK1 was determined by immunoblotting. Data were analyzed with unpaired two-sided Student’s t tests or one-way ANOVA, and presented as mean G SEM, n = 3. *p < 0.05, **p < 0.01.

Article Snippet: Materials purchased from Proteintech Group (Wuhan, China) included antibodies against GAPDH (proteintech, 10494-1-AP, 1:5000), Beta Actin (proteintech, 66009-1-Ig, 1:1000), goat anti-rabbit IgG (proteintech, 66912-1- lg, 1:10000), Pro Caspase-8 (Abcam, ab108333, 1:10000), Caspase-9 (proteintech, 10380-1-AP, 1:1000), Cleaved Caspase-9 (Abcam, ab2324, 1 ug/mL), caspase-3 (proteintech, 19677-1-AP, 1:2000), cleaved caspase-3 (Abcam, ab2302, 1:100), Bcl-2 (proteintech, 12789-1-AP, 1:1000), Bax (proteintech, 50599-2-lg,1:1000), HSP90A (proteintech, 60318-1-Ig, 1:5000), Cdc37 (proteintech, 10218-1-AP, 1:200), CDK1 (proteintech, 19532-1-Ig, 1:200), Cyclin A2 (proteintech, 18202-1-AP, 1:5000).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Expressing, Quantitative RT-PCR, Over Expression

Journal: iScience

Article Title: Bombinin-BO1 induces hepatocellular carcinoma cell-cycle arrest and apoptosis via the HSP90A-Cdc37-CDK1 axis.

doi: 10.1016/j.isci.2024.110382

Figure Lengend Snippet: Figure 8. Schematic diagram of the mechanism by which BO1 regulates HCC BO1 enters the cell through endocytosis and competes with Cdc37 for binding to HSP90A in the cytoplasm, thereby inhibiting the formation of the HSP90A- Cdc37-CDK1 complex and inducing the degradation of CDK1 in both the UPS and ALP modes. Decreased levels of CDK1 induced the occurrence of S-phase cycle block and apoptosis in HCC, which, in turn, inhibited cell proliferative activity. preCDK1, immature CDK1 precursor.

Article Snippet: Materials purchased from Proteintech Group (Wuhan, China) included antibodies against GAPDH (proteintech, 10494-1-AP, 1:5000), Beta Actin (proteintech, 66009-1-Ig, 1:1000), goat anti-rabbit IgG (proteintech, 66912-1- lg, 1:10000), Pro Caspase-8 (Abcam, ab108333, 1:10000), Caspase-9 (proteintech, 10380-1-AP, 1:1000), Cleaved Caspase-9 (Abcam, ab2324, 1 ug/mL), caspase-3 (proteintech, 19677-1-AP, 1:2000), cleaved caspase-3 (Abcam, ab2302, 1:100), Bcl-2 (proteintech, 12789-1-AP, 1:1000), Bax (proteintech, 50599-2-lg,1:1000), HSP90A (proteintech, 60318-1-Ig, 1:5000), Cdc37 (proteintech, 10218-1-AP, 1:200), CDK1 (proteintech, 19532-1-Ig, 1:200), Cyclin A2 (proteintech, 18202-1-AP, 1:5000).

Techniques: Binding Assay, Blocking Assay, Activity Assay

Journal: iScience

Article Title: Bombinin-BO1 induces hepatocellular carcinoma cell-cycle arrest and apoptosis via the HSP90A-Cdc37-CDK1 axis

doi: 10.1016/j.isci.2024.110382

Figure Lengend Snippet: HSP90A serves as the target of BO1 (A) SDS-PAGE analysis of pull-down samples. (B) Venn plot. Samples were analyzed by LC-MS/MS mass spectrometry and screened for proteins specific to the bio-BO1 pull-down samples in combination with FDR<0.01 and Score Sequest HT > 80. (C) The abundance of HSP90A in samples was analyzed by western blot. Input: whole protein from cell lysis, NC: sample pulled by IgG, bio-BO1: sample pulled by bio-BO1. (D and E) The localization of FITC-BO1 and HSP90A in Hep 3B and Huh7 cells was detected by confocal microscopy. green: FITC-BO1, red: HSP90A, yellow: co-localization of FITC-BO1 and HSP90A, scale bar: 20 μm.

Article Snippet: HSP90A , proteintech , Cat#60318-1-Ig; RRID: AB_2881429.

Techniques: SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Lysis, Confocal Microscopy

Journal: iScience

Article Title: Bombinin-BO1 induces hepatocellular carcinoma cell-cycle arrest and apoptosis via the HSP90A-Cdc37-CDK1 axis

doi: 10.1016/j.isci.2024.110382

Figure Lengend Snippet: BO1 induces the degradation of CDKs by regulating HSP90A-Cdc37 (A and B) Putative binding site analysis of BO1 on HSP90A_MC or HSP90A_N and the surrounding amino acid residues by molecular docking. (C and D) Whole-cell lysates from BO1-treated cells were immunoprecipitated and analyzed for the interaction of HSP90A, Cdc37, and CDK1 by immunoblotting. (E) The expression of HSP90A in cells was determined by RT-qPCR. (F and G) Cells were pretreated with 20 μM MG-132 for 4 h before treatment with (+) or without (−) 15 μM BO1 for 24 h, and CDK1 was determined by immunoblotting. (H and I) Overexpression of HSP90A in Hep 3B cells significantly repaired BO1-induced CDK1 reduction. (J and K) Cells were treated with 10 mM NH 4 Cl for 4 h before treatment with (+) or without (−) 15 μM BO1 for 24 h, and then CDK1 was determined by immunoblotting. Data were analyzed with unpaired two-sided Student’s t tests or one-way ANOVA, and presented as mean ± SEM, n = 3. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: HSP90A , proteintech , Cat#60318-1-Ig; RRID: AB_2881429.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Expressing, Quantitative RT-PCR, Over Expression

Journal: iScience

Article Title: Bombinin-BO1 induces hepatocellular carcinoma cell-cycle arrest and apoptosis via the HSP90A-Cdc37-CDK1 axis

doi: 10.1016/j.isci.2024.110382

Figure Lengend Snippet: Schematic diagram of the mechanism by which BO1 regulates HCC BO1 enters the cell through endocytosis and competes with Cdc37 for binding to HSP90A in the cytoplasm, thereby inhibiting the formation of the HSP90A-Cdc37-CDK1 complex and inducing the degradation of CDK1 in both the UPS and ALP modes. Decreased levels of CDK1 induced the occurrence of S-phase cycle block and apoptosis in HCC, which, in turn, inhibited cell proliferative activity. preCDK1, immature CDK1 precursor.

Article Snippet: HSP90A , proteintech , Cat#60318-1-Ig; RRID: AB_2881429.

Techniques: Binding Assay, Blocking Assay, Activity Assay

Journal: iScience

Article Title: Bombinin-BO1 induces hepatocellular carcinoma cell-cycle arrest and apoptosis via the HSP90A-Cdc37-CDK1 axis

doi: 10.1016/j.isci.2024.110382

Figure Lengend Snippet:

Article Snippet: HSP90A , proteintech , Cat#60318-1-Ig; RRID: AB_2881429.

Techniques: Recombinant, Magnetic Beads, RNA Extraction, Quantitation Assay, Protein Concentration, Software, Lysis