primary antibodies rabbit anti-hdac6  (Abnova)

Journal: Frontiers in Oncology

Article Title: MPT0G413, A Novel HDAC6-Selective Inhibitor, and Bortezomib Synergistically Exert Anti-tumor Activity in Multiple Myeloma Cells

doi: 10.3389/fonc.2019.00249

Figure Lengend Snippet: MPT0G413 potently inhibited histone deacetylase (HDAC6) and inhibited multiple myeloma cell growth and proliferation. (A) Chemical structure of MPT0G413. (B) Human multiple myeloma cell lines (RPMI-8226, NCI-H92; density, 1 × 10 4 ) and human bone marrow stromal cells (HS-5; density, 5 × 10 3 ) were incubated with or without the indicated concentrations of MPT0G413 for 48 h. Cell proliferation was evaluated using a 5-bromo-2′-deoxyuridine (BrdU) proliferation assay. (C) RPMI-8226, NCI-H929, and HS-5 cells were exposed to MPT0G413 at the indicated concentrations for 24, 48, and 72 h. Cell viability was measured using a MTT assay. (D,E) RPMI-8226 and NCI-H929 cells were treated with DMSO or MPT0G413 (0.1, 1, 2.5, 10 μM) and SAHA (2.5 μM) for 24 h. Cells were subsequently harvested, and the lysates were subjected to Western blotting of the indicated proteins. Protein levels in the Western blots were quantified using Image J software. The results are shown as mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001, compared with HS-5 cells group (B) and the control group (C–E) .

Article Snippet: We used non-conjugated primary antibodies against HDAC6 (#7612), Caspases-3 (#9661),−8 (#9746), and−9 (#9502), acetyl-histone 3 (#9677), acetyl-histone 4 (#8647), histone 3 (#9715), histone 4 (#2935), acetyl-α-tubulin (#5335), were purchased from Cell Signaling Technology (Danvers, MA, USA). α-tubulin (GTX112141), dynein (GTX80684), ubiquitin (GTX19247), ICAM (GTX100450), LC3B (GTX127375), acetyl-histone 2 (GTX633388) and histone 2 (GTX129418) were purchased from GeneTex (Hsinchu, Taiwan).

Techniques: Histone Deacetylase Assay, Incubation, Proliferation Assay, MTT Assay, Western Blot, Software, Control

Journal: Frontiers in Oncology

Article Title: MPT0G413, A Novel HDAC6-Selective Inhibitor, and Bortezomib Synergistically Exert Anti-tumor Activity in Multiple Myeloma Cells

doi: 10.3389/fonc.2019.00249

Figure Lengend Snippet: MPT0G413 inhibited the binding of histone deacetylase (HDAC6) to dynein and induced the significant accumulation of polyubiquitinated proteins when combined with bortezomib. (A) Hypothetical rationale of proteasome and aggresome pathway inhibition by bortezomib and MPT0G413. (B) RPMI-8226 and NCI-H929 cells were treated with MPT0G413 (1 and 2.5 μM) for 6 h, and total cell lysates were subjected immunoprecipitation with 1 μg of an anti-dynein or anti-ubiquitin antibody, followed by immunoblotting with an anti-HDAC6 antibody. (C) RPMI-8226 and NCI-H929 cells were cultured with MPT0G413 (2.5 μM), bortezomib (2.5 nM), or combined therapy for 12 h. Whole cell lysates were subjected to Western blotting with antibodies specific for ubiquitin and LC3. (D) NCI-H929 cells were treated with MPT0G413 (2.5 μM), bortezomib (2.5 nM), or combination therapy for 6 h and stained by anti-LC3B (green) and anti-ubiquitin (red) antibodies and DAPI (blue) prior to confocal microscopy analysis. Scale bar = 50 μm.

Article Snippet: We used non-conjugated primary antibodies against HDAC6 (#7612), Caspases-3 (#9661),−8 (#9746), and−9 (#9502), acetyl-histone 3 (#9677), acetyl-histone 4 (#8647), histone 3 (#9715), histone 4 (#2935), acetyl-α-tubulin (#5335), were purchased from Cell Signaling Technology (Danvers, MA, USA). α-tubulin (GTX112141), dynein (GTX80684), ubiquitin (GTX19247), ICAM (GTX100450), LC3B (GTX127375), acetyl-histone 2 (GTX633388) and histone 2 (GTX129418) were purchased from GeneTex (Hsinchu, Taiwan).

Techniques: Binding Assay, Histone Deacetylase Assay, Inhibition, Immunoprecipitation, Ubiquitin Proteomics, Western Blot, Cell Culture, Staining, Confocal Microscopy