Journal: Cell communication and signaling : CCS

Article Title: AXL receptor tyrosine kinase modulates gonadotropin-releasing hormone receptor signaling.

doi: 10.1186/s12964-023-01313-y

Figure Lengend Snippet: Fig. 1 AXL and GnRH receptor-like immunoreactivity in murine and human pituitaries. A Representative images showing AXL-like (red) and GnRH receptor-like (green) immunostaining in human, male C57 murine, and C57 female murine pituitary sections. Cell nuclei (blue) blue are shown in the merged images. B Frequency of overlapping AXL- and GnRH receptor-like immunoreactivity in a human pituitary; > 100 cells were analyzed from 7 different images for both AXL- and GnRH receptor-like immunoreactivity. C Representative images showing Gas6-like (red) and GnRH receptor-like (green) immunostaining in human, male C57 murine, and C57 female murine pituitary sections. Cell nuclei (blue) blue are shown in the merged images. D Representative images showing AXL-like (red) and Gas6-like (green) immunostaining in a human pituitary section. Cell nuclei (blue) blue are shown in the merged images. Images are representative of ≥ 3 paraffin sections; scale bars = 20 μm

Article Snippet: After blocking, tissue sections were incubated with GnRHR monoclonal antibody (GNRH03, Thermo Fisher Scientific) at 1:100 dilution, AXL antibody (PA5-77,875, Thermo Fisher Scientific) at 1:200 dilution, AXL antibody (PA5-106,118, Thermo Fisher Scientific) at 1:200 dilution, GnRHR monoclonal antibody (GNRHR/768, ab220196, Abcam) at 1 μg/mL dilution, Gas6 rabbit antibody (A8545, ABclonal) at 1:100 dilution and Gas6 goat antibody (AF885SP, R&D Systems) at 5 μg/mL dilution in blocking buffer in a humidified chamber at 4 °C overnight.

Techniques: Immunostaining

Journal: Cell communication and signaling : CCS

Article Title: AXL receptor tyrosine kinase modulates gonadotropin-releasing hormone receptor signaling.

doi: 10.1186/s12964-023-01313-y

Figure Lengend Snippet: Fig. 3 Gas6-dependent AXL receptor activation enhances GnRH receptor signaling processes. A Proportion of phosphorylated ERK relative to total ERK (pERK/ERK) in αT3-1 and LβT2 cells under control conditions and after incubation with Gas6 (100 nM for 3 h); n = 3 for each condition. B Time course of Gas6-dependent changes (100 nM) in pERK/ERK in LβT2 cells (from left); effect of incubating LβT2 cells with GnRH (10 nM) and GnRH plus Gas6 (100 nM) for 5 min on pERK/ERK (at right); n = 3 for each condition. C Time course of Gas6-dependent (100 nM) changes on Egr-1 transcript abundance in αT3-1 and LβT2 cells; n = 3. D LHβ protein abundance in the culture media and lysates of LβT2 cells under control conditions and after 2 h incubations with Gas6 (100 nM) and GnRH (10 nM); n = 3. € LHβ protein abundance in LβT2 culture media under control conditions and after 2 h incubations with Gas6 (100 nM), GnRH (10 nM), the GnRH receptor antagonist cetrorelix (2 nM), the AXL receptor antagonist R428 (50 µM), and the MEK inhibitor U0126 (10 µM); n = 3 for each condition. n = number of independent experiments; numerical data presented as mean ± SEM; * P < 0.05

Article Snippet: After blocking, tissue sections were incubated with GnRHR monoclonal antibody (GNRH03, Thermo Fisher Scientific) at 1:100 dilution, AXL antibody (PA5-77,875, Thermo Fisher Scientific) at 1:200 dilution, AXL antibody (PA5-106,118, Thermo Fisher Scientific) at 1:200 dilution, GnRHR monoclonal antibody (GNRHR/768, ab220196, Abcam) at 1 μg/mL dilution, Gas6 rabbit antibody (A8545, ABclonal) at 1:100 dilution and Gas6 goat antibody (AF885SP, R&D Systems) at 5 μg/mL dilution in blocking buffer in a humidified chamber at 4 °C overnight.

Techniques: Activation Assay, Control, Incubation, Quantitative Proteomics

Journal: Cell communication and signaling : CCS

Article Title: AXL receptor tyrosine kinase modulates gonadotropin-releasing hormone receptor signaling.

doi: 10.1186/s12964-023-01313-y

Figure Lengend Snippet: Fig. 4 AXL and GnRH receptor stimulation promotes pro-MMP9 release. Pro-MMP9 abundance in the culture media and lysates of αT3-1 (A) and LβT2 (B) cells following incubation with Gas6 (100 nM), GnRH (10 nM), Gas6 plus GnRH, and the AXL receptor antagonist R428 (50 µM), the MEK inhibitor U0126 (10 µM). n = 4 for each condition. n = number of independent experiments; numerical data presented as mean ± SEM; ns = P > 0.05; * P < 0.05

Article Snippet: After blocking, tissue sections were incubated with GnRHR monoclonal antibody (GNRH03, Thermo Fisher Scientific) at 1:100 dilution, AXL antibody (PA5-77,875, Thermo Fisher Scientific) at 1:200 dilution, AXL antibody (PA5-106,118, Thermo Fisher Scientific) at 1:200 dilution, GnRHR monoclonal antibody (GNRHR/768, ab220196, Abcam) at 1 μg/mL dilution, Gas6 rabbit antibody (A8545, ABclonal) at 1:100 dilution and Gas6 goat antibody (AF885SP, R&D Systems) at 5 μg/mL dilution in blocking buffer in a humidified chamber at 4 °C overnight.

Techniques: Incubation

Journal: Cell communication and signaling : CCS

Article Title: AXL receptor tyrosine kinase modulates gonadotropin-releasing hormone receptor signaling.

doi: 10.1186/s12964-023-01313-y

Figure Lengend Snippet: Fig. 5 AXL and GnRH receptor activation promotes migration of clonal gonadotropes. Transwell migratory responses (% cells) of αT3-1 (A) and LβT2 (B) cells following 48 h incubations with Gas6 (100 nM), GnRH (10 nM), Gas6 plus GnRH, the MEK inhibitor U0126 (10 µM), and the MEK activator PAF C-16 (100 µM). n = 4 for each condition. n = number of independent experiments; numerical data presented as mean ± SEM; ns = P > 0.05; * P < 0.05. C Working model of AXL and GnRH receptor signaling integration in gonadotropes

Article Snippet: After blocking, tissue sections were incubated with GnRHR monoclonal antibody (GNRH03, Thermo Fisher Scientific) at 1:100 dilution, AXL antibody (PA5-77,875, Thermo Fisher Scientific) at 1:200 dilution, AXL antibody (PA5-106,118, Thermo Fisher Scientific) at 1:200 dilution, GnRHR monoclonal antibody (GNRHR/768, ab220196, Abcam) at 1 μg/mL dilution, Gas6 rabbit antibody (A8545, ABclonal) at 1:100 dilution and Gas6 goat antibody (AF885SP, R&D Systems) at 5 μg/mL dilution in blocking buffer in a humidified chamber at 4 °C overnight.

Techniques: Activation Assay, Migration