human embryonic kidney cell line hek293t  (ATCC)

Journal: MedComm

Article Title: HSD17B7 Counters Bone Loss in Estrogen Deficiency via Estrogen Receptor Stabilization and Mediates the Effect of Raloxifene

doi: 10.1002/mco2.70623

Figure Lengend Snippet: HSD17B as a binding partner with ERα in preosteoclasts. (A) Primary mouse CD11b + bone marrow cell (BMC) proteins were pulled down by biotin‐labeled ERα followed by proteomics. (B) MS/MS spectra of HSD17B7 peptides bound with ERα are shown. (C) The predicted docking modules of HSD17B7 (blue) and ERα (red) were analyzed by AutoDock and exhibited with PyMol. The docking score was 307 and confidence score 0.9585, indicating a strong potential for real binding. (D) After transfecting HEK293T cells with HA‐ERα or MYC‐HSD17B7, a coimmunoprecipitation assay was performed to determine the physical interaction between HSD17B7 and ERα. (E) Western blot analysis of the protein levels of HSD17B7 and ERα in BMCs from Sham/OVX from young mice; n = 3. (F) Proportions of HSD17B7 and ERα in CD11b + BMCs from Sham/OVX from young mice. Values presented are the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001. Figure was created with Biorender.com.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle medium (DMEM).

Techniques: Binding Assay, Labeling, Tandem Mass Spectroscopy, Co-Immunoprecipitation Assay, Western Blot

Journal: MedComm

Article Title: HSD17B7 Counters Bone Loss in Estrogen Deficiency via Estrogen Receptor Stabilization and Mediates the Effect of Raloxifene

doi: 10.1002/mco2.70623

Figure Lengend Snippet: Blocking the ubiquitin–proteasomal degradation of ERα by HSD17B7. (A) HEK293T cells transfected with ERα were treated with estrogen (E2, 10 nM) for the indicated times, and the relative protein levels of ERα were compared ( n = 3). The blot shown was a representative blot. (B) HEK293T cells were transfected with empty vector or shRNA for HSD17B7. Twenty‐four hours after transfection, the cells were treated with 20 µg/mL cycloheximide (CHX) or 20 µM MG132 for 0.5–2 h. The cell lysates were blotted with GAPDH antibody. The blot shown was a representative blot. (C) HEK293T cells were transfected with ERα and HSD17B7‐OE or shHSD17B7 and then treated with MG132 (20 µM) for 2 h. The cell lysates were immunoblotted with ERα and HSD17B7antibodies, and the results were quantified ( n = 3). The blot shown was a representative blot. (D) HEK293T cells were cotransfected with HSD17B7‐OE or shHSD17B7, and the FasL and ERE luciferase activity in the cell lysates was measured ( n = 4). (E) HEK293T cells were cotransfected with Ub, ERα, and HSD17B7‐OE or shHSD17B7. After 24 h, the cells were treated with E2 (10 nM) for 40 min in the presence of MG132 (2 µM), and total cell lysates were immunoprecipitated with anti‐ERα antibodies and immunoblotted with antiubiquitin antibodies. (F–I) Based on the docking prediction results for HSD17B7 and ERα, potential docking sites were deleted from the HSD17B7‐MYC plasmid. (F) Schemes for deleting N‐terminal sequences of HSD17B7. (G) HEK293T cells were cotransfected with HA‐ERα and WT or deletion‐mutant HSD17B7. Coimmunoprecipitation was performed using anti‐HA or anti‐MYC antibodies, and immunoblots were visualized for HSD17B7 and ERα. (H) HEK293T cells were transfected with WT or deletion‐mutant HSD17B7, and FasL and ERE luciferase activities were measured ( n = 5). (I) HEK293T cells were cotransfected with Ub, ERα, and WT or deletion‐mutant HSD17B7 for 24 h. After 24 h, the cells were treated with E2 (10 nM) for 40 min in the presence of MG132 (2 µM), and total cell lysates were immunoprecipitated using anti‐ERα antibody and immunoblotted using anti‐Ub antibody. Values presented are the mean ± SEM. * p < 0.05 versus EV.

Article Snippet: Human embryonic kidney 293T (HEK293T) cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle medium (DMEM).

Techniques: Blocking Assay, Ubiquitin Proteomics, Transfection, Plasmid Preparation, shRNA, Luciferase, Activity Assay, Immunoprecipitation, Mutagenesis, Western Blot