plasmid encoding erα  (Addgene inc)

Journal: bioRxiv

Article Title: Antidepressants interact with sex steroid receptors and their intracellular signaling components

doi: 10.64898/2026.03.17.712321

Figure Lengend Snippet: In situ PLA demonstrates close proximity between biotinylated fluoxetine as well as HNK and ERα (red dots) in MCF-7 cells and transfected HEK293T cells (magenta), while no PLA signal was detected in HEK293T cells not expressing ERα. ERα-transfected cells are tagged with GFP and are represented in magenta. Blue represents DAPI staining. Arrows point to the PLA signal.

Article Snippet: A group of HEK293T cells were transfected with a plasmid encoding ERα (pEGFP-C1-ER alpha, Addgene, Plasmid #28230) using Lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: In Situ, Transfection, Expressing, Staining

Journal: Nature microbiology

Article Title: Nuclear pore blockade reveals that HIV-1 completes reverse transcription and uncoating in the nucleus.

doi: 10.1038/s41564-020-0735-8

Figure Lengend Snippet: Fig. 1 | Artificial NPC blockade inhibits HIV-1 infection at the nuclear entry stage. a, Schematics of the Nup62 construct fused to DmrB and two copies of eGFP (Nup62DG) used to block active nuclear pore transport after the addition of B/B HD. b, HeLa cells were stably expressing the Nup62DG construct and were transfected with ER-α fused to mCherry. Twenty-four hours post-transfection, cells were treated with E2 for 30 min in the presence or absence of HD. The efficiency of nuclear pore blockade was quantified by counting cells having either a nuclear ER-α signal (less efficient) or both nuclear and cytoplasmic ER-α signals (efficient nuclear pore block). The data shown are representative of three independent experiments. c, Mock or Nup62DG-transduced HeLa, THP1-differentiated macrophages, CEM and SupT1 T cells were synchronously infected with VSV-G pseudotyped R7ΔEnvmCherry in the presence or absence of HD drug for the first 24 h of infection. The HD drug was removed after 24 h, replaced with normal media and infection was assessed 48 h post-synchronized infection by measuring the percentage of mCherry+ cells. The normalized and averaged data (± s.e.m.) from three independent experiments are shown. d, RT–qPCR quantification of reverse transcription and 2-LTR circles in cells expressing Nup62DG, 24 h after HIV-1 infection. Depicted is the mean of biological triplicates (± s.d.). Data shown are representative of three independent experiments. e, HeLa and THP1-differentiated macrophages that stably express the Nup62DG construct were synchronously infected with VSV-G-R7ΔEnvmCherry. Cells were treated with the HD drug for 4 h, then fixed and stained for HIV-1 capsid protein; p24 (red) and DAPI (blue) for the cell nucleus. Colocalization between Nup62DG with p24 (boxed region) is shown by the arrows. f,g, We employed a quantification process as described in the Methods and Extended Data Fig. 2, to detect perinuclear and nuclear p24 protein levels in the HeLa- (f) and THP1-differentiated (g) macrophages described in e. Twenty or more cells were analysed in each experiment. Data were averaged (± s.e.m.) from three independent experiments. Statistical significance was assessed using a two-way ANOVA and Bonferroni post-test. P < 0.05 was considered significant.

Article Snippet: ERα fused to mCherry was generated by cloning the ERα open reading frame (plasmid no. 28230; Addgene) into the pLVX-mCherry-C1 backbone (Clontech).

Techniques: Infection, Construct, Blocking Assay, Stable Transfection, Expressing, Transfection, Quantitative RT-PCR, Reverse Transcription, Staining