Journal: Molecular Vision

Article Title: Melatonin receptor heterodimerization in a photoreceptor-like cell line endogenously expressing melatonin receptors

doi:

Figure Lengend Snippet: MT 1 and MT 2 form heterodimers in 661W cells. Upper panels: Immunofluorescence for melatonin receptor type 1 (MT 1 ) and MT 2 receptors and colocalization. Scale bars = 100 µm. Lower panels: Proximity ligation assay (PLA) showing the MT 1 /MT 2 interaction (positive signal shown as red dots in the right panel). Cell nuclei are shown in green. Scale bars: left and center = 100 µm, right = 50 µm.

Article Snippet: Cells were directly washed in PBS (1X; KCl 2.67 mM, KH 2 PO 4 1.47 mM, NaCl 139.9 mM, Na 2 HPO 4 , 8.1 mM, pH 7.4) and fixed in cold acetone for 2 min. Once fixed, the cells were washed in PBS twice for 10 min and incubated in BLOXALL (Vector Laboratories, Burlingame, CA) to block endogenous peroxidases and phosphatases for 10 min. Then, samples were washed twice with PBS for 10 min, blocked with PBS containing 0.02% Triton and 5% bovine serum albumin (BSA; 1 h at room temperature), and incubated with the primary antibodies MT 1 (0.004 mg/ml; rabbit, AMR-031; Alomone Laboratories, Jerusalem, Israel) and MT 2 (0.004 mg/ml; goat, SC13177; Santa Cruz Biotechnology, Santa Cruz, CA) for 3 h at room temperature.

Techniques: Immunofluorescence, Proximity Ligation Assay

Journal: Molecular Vision

Article Title: Melatonin receptor heterodimerization in a photoreceptor-like cell line endogenously expressing melatonin receptors

doi:

Figure Lengend Snippet: Genetic ablation of MT 2 with CRISPR/Cas9 system does not affect MT 1 mRNA or protein. Top panels: cDNA from 661W and 661W-MT 2 −/− cells was analyzed with reverse transcriptase PCR (RT–PCR) to detect MT 1 (left) and MT 2 (right) expression. Middle panels: Immunofluorescence was performed in 661W and 661W-melatonin receptor type 2 (MT 2 ) −/− cells to detect MT 1 (left) and MT 2 (right) proteins. The positive signal of each receptor is shown in green, and nuclei are counterstained in red. Lower panel: The presence or absence of MT 1 /MT 2 heterodimer in 661W and 661W-MT 2 −/− cells was assessed with proximity ligation assay (PLA; positive signal in red), and nuclei were counterstained in green. MT 1 expression (transcript and protein) was detected in 661W and 661W-MT 2 −/− cells, whereas MT 2 expression (transcript and protein) was detected only in 661W cells. Accordingly, MT 1/2h was found only in 661W cells. Scale bars = 100 µm.

Article Snippet: Cells were directly washed in PBS (1X; KCl 2.67 mM, KH 2 PO 4 1.47 mM, NaCl 139.9 mM, Na 2 HPO 4 , 8.1 mM, pH 7.4) and fixed in cold acetone for 2 min. Once fixed, the cells were washed in PBS twice for 10 min and incubated in BLOXALL (Vector Laboratories, Burlingame, CA) to block endogenous peroxidases and phosphatases for 10 min. Then, samples were washed twice with PBS for 10 min, blocked with PBS containing 0.02% Triton and 5% bovine serum albumin (BSA; 1 h at room temperature), and incubated with the primary antibodies MT 1 (0.004 mg/ml; rabbit, AMR-031; Alomone Laboratories, Jerusalem, Israel) and MT 2 (0.004 mg/ml; goat, SC13177; Santa Cruz Biotechnology, Santa Cruz, CA) for 3 h at room temperature.

Techniques: CRISPR, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence, Proximity Ligation Assay, Polymerase Chain Reaction

Journal: Molecular Vision

Article Title: Melatonin receptor heterodimerization in a photoreceptor-like cell line endogenously expressing melatonin receptors

doi:

Figure Lengend Snippet: MEL and IIK7 inhibit forskolin-induced cAMP production only in the presence of MT 1/2h . A : Effect of melatonin (MEL) on forskolin-induced cAMP production in 661W cells. B : IIK7 effect on forskolin-induced cAMP production in 661W cells. C : Effect of luzindole (LUZ) on the effect of MEL on forskolin-induced cAMP production. D : Effect of MEL on forskolin-cAMP in 661W-melatonin receptor type 2 (MT 2 ) −/− cells. In all the cases, results (n = 6) are shown as mean ± standard error of the mean (SEM). Asterisks indicate statistically significant differences compared to the control group with one-way ANOVA (Student–Newman–Keuls (SNK) post-hoc test; p<0.05).

Article Snippet: Cells were directly washed in PBS (1X; KCl 2.67 mM, KH 2 PO 4 1.47 mM, NaCl 139.9 mM, Na 2 HPO 4 , 8.1 mM, pH 7.4) and fixed in cold acetone for 2 min. Once fixed, the cells were washed in PBS twice for 10 min and incubated in BLOXALL (Vector Laboratories, Burlingame, CA) to block endogenous peroxidases and phosphatases for 10 min. Then, samples were washed twice with PBS for 10 min, blocked with PBS containing 0.02% Triton and 5% bovine serum albumin (BSA; 1 h at room temperature), and incubated with the primary antibodies MT 1 (0.004 mg/ml; rabbit, AMR-031; Alomone Laboratories, Jerusalem, Israel) and MT 2 (0.004 mg/ml; goat, SC13177; Santa Cruz Biotechnology, Santa Cruz, CA) for 3 h at room temperature.

Techniques: Control

Journal: Molecular Vision

Article Title: Melatonin receptor heterodimerization in a photoreceptor-like cell line endogenously expressing melatonin receptors

doi:

Figure Lengend Snippet: MEL activates AKT/FoxO1 and ERK1/2 only in the presence of the MT 1/2h . A : Effect of melatonin (MEL; 100 nM) or IIK7 (1,000 nM) 90-min treatment on the phosphorylation of AKT and FoxO1 in 661W cells and 661W-melatonin receptor type 2(MT 2 ) −/− cells. B : Effect of MEL (100 nM) or IIK7 (1,000 nM) 90-min treatment on the phosphorylation of ERK1/2 in 661W cells and 661W-MT 2 −/− cells. In all cases, results (n = 5) are shown as mean ± standard error of the mean (SEM). Asterisks indicate statistically significant differences compared to the control group with one-way ANOVA (Student–Newman–Keuls (SNK) post-hoc test; p<0.05).

Article Snippet: Cells were directly washed in PBS (1X; KCl 2.67 mM, KH 2 PO 4 1.47 mM, NaCl 139.9 mM, Na 2 HPO 4 , 8.1 mM, pH 7.4) and fixed in cold acetone for 2 min. Once fixed, the cells were washed in PBS twice for 10 min and incubated in BLOXALL (Vector Laboratories, Burlingame, CA) to block endogenous peroxidases and phosphatases for 10 min. Then, samples were washed twice with PBS for 10 min, blocked with PBS containing 0.02% Triton and 5% bovine serum albumin (BSA; 1 h at room temperature), and incubated with the primary antibodies MT 1 (0.004 mg/ml; rabbit, AMR-031; Alomone Laboratories, Jerusalem, Israel) and MT 2 (0.004 mg/ml; goat, SC13177; Santa Cruz Biotechnology, Santa Cruz, CA) for 3 h at room temperature.

Techniques: Phospho-proteomics, Control