phosphorylated form  (Cell Signaling Technology Inc)

Journal: Biomedicines

Article Title: Secosteroid–2-Pyrazoline Hybrids: Design, Synthesis, Biological Evaluation and Development of Therapeutic Combinations Against ERα-Positive Breast Cancer Cells

doi: 10.3390/biomedicines13123057

Figure Lengend Snippet: Effect of lead secosteroid–2-pyrazoline hybrids 3f , 3j , and 3k on signaling pathways in MCF-7 breast cancer cells. Antibodies against androgen receptor (AR), estrogen receptor α (ERα), GREB1, S6K, and its phosphorylated form (p-S6K), Bcl-2, and its phosphorylated form (p-Bcl-2), and α-tubulin were used for immunoblotting.

Article Snippet: Primary antibodies against androgen receptor (AR), estrogen receptor α (ERα), GREB1, S6K, and its phosphorylated form (p-S6K), Bcl-2, and its phosphorylated form (p-Bcl-2), and α-tubulin were employed (Cell Signaling Technology, Danvers, MA, USA). α-Tubulin served as the internal loading control.

Techniques: Protein-Protein interactions, Western Blot

Journal: Scientific Reports

Article Title: Somatostatin regulates the clock sensitivity to evening light

doi: 10.1038/s41598-025-26904-2

Figure Lengend Snippet: SST m/m mice show higher light sensitivity at dusk in DD condition. ( A ), 200-lx light pulse at CT12 induced larger phase delays in SST m/m mice. Left , Representative double-plotted actograms of SST +/+ ( Upper ) or SST m/m mice ( Lower) illuminated with a light pulse (200 lx, 30 min) at CT12. Yellow boxes indicate the time of light illumination. Right , Amounts of phase delay (mean ± s.e.m; n = 10 ( SST +/+ ) and 9 ( SST m/m ); ** P < 0.01, unpaired t -test). ( B ), Effect of dim light pulse (2 lx, 30 min) at CT12. Left , Representative double-plotted actograms of SST +/+ ( Upper ) or SST m/m mice ( Lower ). Yellow boxes indicate the time of light illumination. Middle, Magnified presentations of the activities shown in left panels. Right, Amounts of phase delay (mean ± s.e.m; n = 10 ( SST +/+ ) and 11 ( SST m/m ); *** P < 0.001, unpaired t -test). ( C) , Dim light pulse (2 lx, 30 min) at CT12 induced higher cFos mRNA expression and ERK phosphorylation in the SCN of SST m/m mice. Expression of cFos mRNA was examined by in situ hybridization using digoxigenin-labelled probe. Phosphorylated ERK was immunohistochemically stained with pERK antibody. Representative micrographs of cFos mRNA expression ( Left panels ) and phosphorylated ERK ( Right panels ). Arrows indicate the cFos mRNA positive cells and phosphorylated ERK-positive cells. Scale bars, 100 μm. ( D ), Confocal microscopy revealed abundant cells (DAPI, blue) in the VL SCN surrounded by both RHT (green) and SST (red) terminals in SST +/+ mice. Note the dense distribution of SST-terminals proximal to the RHT-terminals in the VL SCN of SST +/+ mice, but none in SST m/m mice. Scale bars, 100 μm ( Upper ) and 10 μm ( Lower ). ( E ), Exposure to a dim light pulse (2 lx, 30 min) at CT12 induced cFOS protein expression in SSTR1 -positive cells in the VL SCN of SST m/m mice. Left , Expression of SSTR1 mRNA (blue) and cFOS protein (brown) was examined. The lower panels show a magnified image of the area indicated by the red rectangle displayed in the upper panels. Arrowheads indicate the cells expressing both SSTR1 mRNA and cFOS protein. Right , Ratio of cFOS-positive cells in SSTR1 -positive cells (mean ± s.e.m; n = 4 (both genotypes); ** P < 0.01, unpaired t -test). Scale bars, 100 μm ( Upper ) and 10 μm ( Lower ). ( F ), Electron microscopy revealed that SST neurons innervate cells in the VL SCN. Left , asymmetrical axo-dendritic input (red arrow) from SST-negative axon to SST-positive dendrite. N, nucleus. Right , symmetrical axo-somatic output (innervation: blue arrows) from SST-positive axon to SST-negative soma. Scale bars, 500 nm. ( G ), Increased SSTR1 mRNA expression in the SCN of SST m/m mice. Quantitative RT-PCR analysis of SSTR1 mRNA was performed using SCN tissue samples precisely collected from SST m/m and SST +/+ mice using a laser-microdissection technique. ( H ), CH-275, an SSTR1 agonist, ameliorated the abnormal delay in locomotor activity in SST m/m mice under long photoperiod. Representative double-plotted actograms of vehicle- ( upper ) or CH-275-treated SST m/m mice ( lower ) entrained for more than three weeks in LD18h:6 h and released to DD at ZT12. CH-275 was administered into the SCN of SST m/m mice using an osmotic mini pump. The time of the surgery is indicated by the stars. Amounts of phase advance (mean ± s.e.m; n = 6 (vehicle) and 7 (CH-275); * P < 0.05, unpaired t -test). The phase was determined by fitting a regression line across several days in DD and extrapolating it back to the first day.

Article Snippet: We used a rabbit polyclonal antibody specific for the phosphorylated forms of ERK 1 and 2 (Cell Signaling; catalog code 9101) and a rabbit antibody for cFOS protein identification (Abcam; catalog code 7963).

Techniques: Expressing, Phospho-proteomics, In Situ Hybridization, Staining, Confocal Microscopy, Electron Microscopy, Quantitative RT-PCR, Laser Capture Microdissection, Activity Assay