flag-tagged ar plasmids with lbd mutations  (Addgene inc)

Journal: Cancer Research

Article Title: An Autophagy-Targeting Chimera Induces Degradation of Androgen Receptor Mutants and AR-v7 in Castration-Resistant Prostate Cancer

doi: 10.1158/0008-5472.CAN-24-0591

Figure Lengend Snippet: ATC-324 degrades AR-LBD mutants and AR-v7/AR-FL heterodimer. A, Immunoblot analysis of AR with or without LBD mutation (L702H, F877L, T878A, M896V, and H874Y) in PC3 cells transfected with the indicated plasmid and treated with ATC-324 (5 µmol/L, 24 hours). The levels of β-actin were examined as a loading control. B and C, Immunoblot analysis of AR-FL and AR-v7 in 22Rv1 cells treated with ATC-324 at the indicated concentrations for 24 hours ( B ) and densitometry of B ( C ). The protein levels were normalized with β-actin. D and E, Fluorescence images of the PLA of FLAG and p62 in PC3 cells ( D ) and quantification of the number of red puncta ( E ). The cells were transfected with untagged AR-FL and FLAG-AR-v7 plasmids and treated with indicated chemicals (5 µmol/L, 12 hours). n >30 cells. F, Immunoblot analysis of FLAG in PC3 cells transfected with indicated plasmids and treated with or without ATC-324 (5 µmol/L, 24 hours). The protein levels were normalized to β-actin and the densitometry values of FLAG corresponding to AR-FL and AR-v7 were labeled below the panels. G, Immunoblot analysis of FLAG in PC3 cells transfected with indicated plasmids and treated with or without ATC-324 (5 µmol/L, 24 hours). The protein levels were normalized to β-actin and the densitometry values of FLAG corresponding to AR-FL and AR-v7 were labeled below the panels. Data are shown as means ± SEM ( n = 3 independent experiments), and statistical significance was analyzed using one-way ANOVA, followed by Tukey post hoc procedures.

Article Snippet: Flag-tagged AR plasmids with LBD mutations were constructed utilizing Flag-M4-AR (RRID: Addgene plasmid_171240), a gift from Steven Balk (Harvard Medical School), via site-directed mutagenesis.

Techniques: Western Blot, Mutagenesis, Transfection, Plasmid Preparation, Control, Fluorescence, Labeling

Journal: FEBS letters

Article Title: Heteromerization between α 1B -adrenoceptor and chemokine (C-C motif) receptor 2 biases α 1B -adrenoceptor signaling: Implications for vascular function.

doi: 10.1002/1873-3468.14463

Figure Lengend Snippet: Fig. 1. Recombinant and vascular smooth muscle CCR2 form heteromers with a1b/B-AR. (A,B) BRET between CCR2 and a1b-AR in HEK293T cells. Cells were transfected with a fixed amount of a1b-AR-RLuc and increasing amounts of CCR2-YFP (circles), mGlu1R-YFP (squares), or YFP (triangles) (A), or with a fixed amount of CCR2-RLuc and increasing amounts of a1b-AR-YFP (circles), mGlu1R-YFP (squares), or YFP (tri- angles) (B). Forty-eight hours after transfection, YFP fluorescence and luminescence were read as described in Materials and methods. Net BRET (528/460 nm) was plotted against YFP fluorescence/luminescence (YFP/Lum). The graphs are representative of three independent experiments. (C) PLA to detect (top to bottom) CCR2, a1B-AR and CCR2:a1B-AR heteromers in hVSMCs (center) and human mesenteric arteries (right). As controls, cells were incubated with mouse or rabbit IgG (m/rIgG) (left). Images show merged 40,6-diamidino-2- phenylindole (DAPI, nuclear counterstain) and PLA signals (red, kexcitation/emission 598/634 nm) and are representative of n = 3 indepen- dent experiments with hVSMCs and human arteries from 3 different donors. Scale bars: left and center – 20 lm; right – 0.5 mm.

Article Snippet: FLAG-tagged Tango plasmid a1b-AR-TANGO (#66214) was from Addgene (Watertown, MA, USA) deposited by the laboratory of Bryan Roth.

Techniques: Recombinant, Transfection, Incubation

Journal: FEBS letters

Article Title: Heteromerization between α 1B -adrenoceptor and chemokine (C-C motif) receptor 2 biases α 1B -adrenoceptor signaling: Implications for vascular function.

doi: 10.1002/1873-3468.14463

Figure Lengend Snippet: Fig. 3. Ligand binding to CCR2 cross recruits b-arrestin to ligand free a1b-AR and increases b-arrestin recruitment to activated a1b-AR. (A) Flow cytometry for the detection of FLAG-a1b-AR-Tango with anti-FLAG (top) and of HA-CCR2 with anti-HA (bottom). HTLA cells were trans- fected with FLAG-a1b-AR-Tango plus pcDNA3 (red line) or HA-CCR2 (green line). Gray area: unstained cells. (B,C) a1b-AR PRESTO-Tango b- arrestin recruitment assays. HTLA cells were transfected with a1b-AR-Tango plus pcDNA3 (circles) or with a1b-AR-Tango plus CCR2 (squares), as in (A). RLU (%): relative luminescence units (RLU) subtracted by the RLU of unstimulated cells and expressed as % RLU mea- sured in cells transfected with a1b-AR-Tango plus pcDNA3 and stimulated with the highest concentration of phenylephrine (PE). (B) Cells were stimulated with increasing concentrations of PE (open symbols) or CCL2 (black symbols). (C) Cells were stimulated with increasing concentrations of PE plus vehicle (open symbols) or 100 nM CCL2 (black symbols).

Article Snippet: FLAG-tagged Tango plasmid a1b-AR-TANGO (#66214) was from Addgene (Watertown, MA, USA) deposited by the laboratory of Bryan Roth.

Techniques: Ligand Binding Assay, Flow Cytometry, Transfection, Concentration Assay

Journal: FEBS letters

Article Title: Heteromerization between α 1B -adrenoceptor and chemokine (C-C motif) receptor 2 biases α 1B -adrenoceptor signaling: Implications for vascular function.

doi: 10.1002/1873-3468.14463

Figure Lengend Snippet: Fig. 4. CCL2/CCR2 regulate Gaq signaling and internalization of a1B-AR from the CCR2:a1B-AR heteromer in hVSMCs. (A–C) hVSMCs were transfected with nontarget-siRNA (NT siRNA) or CCR2 siRNA. (A) PLA to visualize individual receptors and receptor interactions, as in Fig. 1C. Images show merged 40,6-diamidino-2-phenylindole (DAPI, nuclear counterstain) and PLA signals (red, kexcitation/emission 598/ 634 nm) and are representative of n = 3 independent experiments. Scale bars: 20 lm. (B) Quantification of PLA signals from n = 3 indepen- dent experiments. PLA signals (% NT siRNA): PLA signals in % of PLA signals in cells incubated with NT siRNA (=100%). *: P < 0.05 vs. cells incubated with NT siRNA. (C) hVSMCs were treated with vehicle, 1 lM PE, 200 nM CCL2 or both for 5 min. IP3 production was mea- sured by ELISA. N = 3 independent experiments. *: P < 0.05 vs. cells incubated with NT siRNA and stimulated with vehicle. #: P < 0.05 vs. cells incubated with NT siRNA and stimulated with PE alone. (D,E) hVSMCs were stimulated with 200 nM CCL2 at 37 °C for 30 min. (D) Flow cytometry to detect CCR2 (top) and a1B-AR (bottom). Red lines: unstimulated cells. Green lines: cells after CCL2 treatment. Gray area: unstained cells. (E) Quantification of receptor expression levels from n = 3 independent experiments, as in (D). Receptor expression is expressed as % of unstimulated cells. *: P < 0.05 vs. unstimulated cells.

Article Snippet: FLAG-tagged Tango plasmid a1b-AR-TANGO (#66214) was from Addgene (Watertown, MA, USA) deposited by the laboratory of Bryan Roth.

Techniques: Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing