Journal: Pharmaceuticals

Article Title: Histamine H 1 Receptor-Mediated CREB Phosphorylation via G q Protein Signaling and Arrestin Modulation

doi: 10.3390/ph19020227

Figure Lengend Snippet: Concentration-dependent effects of histamine on CREB phosphorylation. CHO cells expressing WT ( A ), S487Trunc ( B ), and S487A ( C ) mutant H 1 receptors were stimulated with or without (control) the indicated concentrations of histamine for 30 min, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are presented in the upper panels. Arrowheads indicate phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control in the lower graphs. Values represent the mean ± SE of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s test). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

Article Snippet: The membranes were then incubated with primary antibodies against CREB (1:4000; #4820, Cell Signaling Technology, Danvers, MA, USA), phosphorylated CREB (1:4000; #9198, Cell Signaling Technology), β-arrestin1 (1:4000; ab32096, Abcam), and β-arrestin2 (1:4000; ab54790, Abcam) for 1 h and then reacted with peroxidase-conjugated secondary antibody (1:4000; #12-348 or #12-349, MilliporeSigma) for 30 min. After detecting CREB or arrestins, antibodies were stripped by incubating the membranes with WB Stripping Solution (Nacalai Tesque) for 5 min, and the membranes were washed with Tris-buffered saline with Tween 20 (Nacalai Tesque).

Techniques: Concentration Assay, Phospho-proteomics, Expressing, Mutagenesis, Control, Western Blot, Binding Assay

Journal: Pharmaceuticals

Article Title: Histamine H 1 Receptor-Mediated CREB Phosphorylation via G q Protein Signaling and Arrestin Modulation

doi: 10.3390/ph19020227

Figure Lengend Snippet: Effects of histamine on CREB phosphorylation in original CHO-K1 cells. CHO-K1 cells were stimulated with or without (control) 100 μM histamine for 30 min, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are presented in the upper panels. The arrowhead indicates phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control in the lower graphs. Values represent the mean ± SE of four independent experiments. CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

Article Snippet: The membranes were then incubated with primary antibodies against CREB (1:4000; #4820, Cell Signaling Technology, Danvers, MA, USA), phosphorylated CREB (1:4000; #9198, Cell Signaling Technology), β-arrestin1 (1:4000; ab32096, Abcam), and β-arrestin2 (1:4000; ab54790, Abcam) for 1 h and then reacted with peroxidase-conjugated secondary antibody (1:4000; #12-348 or #12-349, MilliporeSigma) for 30 min. After detecting CREB or arrestins, antibodies were stripped by incubating the membranes with WB Stripping Solution (Nacalai Tesque) for 5 min, and the membranes were washed with Tris-buffered saline with Tween 20 (Nacalai Tesque).

Techniques: Phospho-proteomics, Control, Western Blot, Binding Assay

Journal: Pharmaceuticals

Article Title: Histamine H 1 Receptor-Mediated CREB Phosphorylation via G q Protein Signaling and Arrestin Modulation

doi: 10.3390/ph19020227

Figure Lengend Snippet: Time-dependent effects of histamine on CREB phosphorylation. CHO cells expressing WT ( A ), S487Trunc ( B ), and S487A ( C ) mutant H 1 receptors were stimulated with or without (control) 100 µM histamine for 10–360 min, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are depicted in the upper panels ( A – C ). Arrowheads indicate phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control in the lower graphs. Values represent the mean ± SE of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s test). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

Article Snippet: The membranes were then incubated with primary antibodies against CREB (1:4000; #4820, Cell Signaling Technology, Danvers, MA, USA), phosphorylated CREB (1:4000; #9198, Cell Signaling Technology), β-arrestin1 (1:4000; ab32096, Abcam), and β-arrestin2 (1:4000; ab54790, Abcam) for 1 h and then reacted with peroxidase-conjugated secondary antibody (1:4000; #12-348 or #12-349, MilliporeSigma) for 30 min. After detecting CREB or arrestins, antibodies were stripped by incubating the membranes with WB Stripping Solution (Nacalai Tesque) for 5 min, and the membranes were washed with Tris-buffered saline with Tween 20 (Nacalai Tesque).

Techniques: Phospho-proteomics, Expressing, Mutagenesis, Control, Western Blot, Binding Assay

Journal: Pharmaceuticals

Article Title: Histamine H 1 Receptor-Mediated CREB Phosphorylation via G q Protein Signaling and Arrestin Modulation

doi: 10.3390/ph19020227

Figure Lengend Snippet: Effects of GRK, clathrin, and dynamin inhibitors on CREB phosphorylation. CHO cells expressing WT H 1 receptors were stimulated with or without 100 µM histamine for 10 min in the presence or absence (vehicle) of inhibitors against GRK2/3 (cmpd101; 30 μM), clathrin (a high concentration of sucrose; 0.32 M), and dynamin (dynasore; 100 μM), and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are depicted in the upper panels. An arrowhead indicates phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the vehicle control without histamine in the lower graphs. Values represent the mean ± SE of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. vehicle without histamine treatment (one-way ANOVA followed by Tukey’s test). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type; GRK, G protein-coupled receptor kinase.

Article Snippet: The membranes were then incubated with primary antibodies against CREB (1:4000; #4820, Cell Signaling Technology, Danvers, MA, USA), phosphorylated CREB (1:4000; #9198, Cell Signaling Technology), β-arrestin1 (1:4000; ab32096, Abcam), and β-arrestin2 (1:4000; ab54790, Abcam) for 1 h and then reacted with peroxidase-conjugated secondary antibody (1:4000; #12-348 or #12-349, MilliporeSigma) for 30 min. After detecting CREB or arrestins, antibodies were stripped by incubating the membranes with WB Stripping Solution (Nacalai Tesque) for 5 min, and the membranes were washed with Tris-buffered saline with Tween 20 (Nacalai Tesque).

Techniques: Phospho-proteomics, Expressing, Concentration Assay, Western Blot, Control, Binding Assay

Journal: Pharmaceuticals

Article Title: Histamine H 1 Receptor-Mediated CREB Phosphorylation via G q Protein Signaling and Arrestin Modulation

doi: 10.3390/ph19020227

Figure Lengend Snippet: Effects of histamine H 1 receptor antagonist on CREB phosphorylation. CHO cells expressing WT ( A ) and S487Trunc ( B ) mutant H 1 receptors were stimulated with or without 100 µM histamine for 10 min in the presence or absence (vehicle) of histamine H 1 receptor antagonist ketotifen (1000 μM), and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are illustrated in the upper panels ( A , B ). Arrowheads indicate phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the vehicle control without histamine in the lower graphs ( A , B ). Values represent the mean ± SE of four independent experiments. ** p < 0.01, *** p < 0.001 vs. vehicle without histamine, ## p < 0.01, ### p < 0.001 vs. vehicle with histamine (one-way ANOVA followed by Tukey’s test). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

Article Snippet: The membranes were then incubated with primary antibodies against CREB (1:4000; #4820, Cell Signaling Technology, Danvers, MA, USA), phosphorylated CREB (1:4000; #9198, Cell Signaling Technology), β-arrestin1 (1:4000; ab32096, Abcam), and β-arrestin2 (1:4000; ab54790, Abcam) for 1 h and then reacted with peroxidase-conjugated secondary antibody (1:4000; #12-348 or #12-349, MilliporeSigma) for 30 min. After detecting CREB or arrestins, antibodies were stripped by incubating the membranes with WB Stripping Solution (Nacalai Tesque) for 5 min, and the membranes were washed with Tris-buffered saline with Tween 20 (Nacalai Tesque).

Techniques: Phospho-proteomics, Expressing, Mutagenesis, Western Blot, Control, Binding Assay

Journal: Pharmaceuticals

Article Title: Histamine H 1 Receptor-Mediated CREB Phosphorylation via G q Protein Signaling and Arrestin Modulation

doi: 10.3390/ph19020227

Figure Lengend Snippet: Effects of a G q protein inhibitor, intracellular Ca 2+ chelator, and PKC inhibitor on CREB phosphorylation. CHO cells expressing WT ( A , C ) and S487Trunc mutant ( B , D ) H 1 receptors were stimulated with or without 100 µM histamine for 10 min in the presence or absence (vehicle) of the G q protein inhibitor YM-254890 (20 μM), intracellular Ca 2+ chelator BAPTA-AM (50 μM), or PKC inhibitor GF109203X (10 μM). Protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are depicted in the upper panels ( A – D ). Arrowheads indicate phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the vehicle control without histamine in the lower graphs ( A – D ). Values represent the mean ± SE of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.01 vs. vehicle without histamine, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. vehicle with histamine (one-way ANOVA followed by Tukey’s test). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type; PKC, Protein kinase C.

Article Snippet: The membranes were then incubated with primary antibodies against CREB (1:4000; #4820, Cell Signaling Technology, Danvers, MA, USA), phosphorylated CREB (1:4000; #9198, Cell Signaling Technology), β-arrestin1 (1:4000; ab32096, Abcam), and β-arrestin2 (1:4000; ab54790, Abcam) for 1 h and then reacted with peroxidase-conjugated secondary antibody (1:4000; #12-348 or #12-349, MilliporeSigma) for 30 min. After detecting CREB or arrestins, antibodies were stripped by incubating the membranes with WB Stripping Solution (Nacalai Tesque) for 5 min, and the membranes were washed with Tris-buffered saline with Tween 20 (Nacalai Tesque).

Techniques: Phospho-proteomics, Expressing, Mutagenesis, Western Blot, Control, Binding Assay

Journal: Pharmaceuticals

Article Title: Histamine H 1 Receptor-Mediated CREB Phosphorylation via G q Protein Signaling and Arrestin Modulation

doi: 10.3390/ph19020227

Figure Lengend Snippet: Effects of ERK and JNK inhibitors on CREB phosphorylation. CHO cells expressing WT ( A , C ) and S487Trunc mutant ( B , D ) H 1 receptors were stimulated with or without 100 µM histamine for 10 min in the presence or absence (vehicle) of the ERK inhibitor SCH772984 (20 μM) or the JNK inhibitor SP600125 (20 μM), and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are depicted in the upper panels ( A – D ). Arrowheads indicate phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the vehicle control without histamine in the lower graphs ( A – D ). Values represent the mean ± SE of four independent experiments. ** p < 0.01, *** p < 0.001 vs. vehicle without histamine. ## p < 0.01, ### p < 0.001 vs. vehicle with histamine (one-way ANOVA followed by Tukey’s test). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

Article Snippet: The membranes were then incubated with primary antibodies against CREB (1:4000; #4820, Cell Signaling Technology, Danvers, MA, USA), phosphorylated CREB (1:4000; #9198, Cell Signaling Technology), β-arrestin1 (1:4000; ab32096, Abcam), and β-arrestin2 (1:4000; ab54790, Abcam) for 1 h and then reacted with peroxidase-conjugated secondary antibody (1:4000; #12-348 or #12-349, MilliporeSigma) for 30 min. After detecting CREB or arrestins, antibodies were stripped by incubating the membranes with WB Stripping Solution (Nacalai Tesque) for 5 min, and the membranes were washed with Tris-buffered saline with Tween 20 (Nacalai Tesque).

Techniques: Phospho-proteomics, Expressing, Mutagenesis, Western Blot, Control, Binding Assay

Journal: Pharmaceuticals

Article Title: Histamine H 1 Receptor-Mediated CREB Phosphorylation via G q Protein Signaling and Arrestin Modulation

doi: 10.3390/ph19020227

Figure Lengend Snippet: Effects of arrestin overexpression on CREB phosphorylation. ( A ) β-arrestin1 or β-arrestin2 was overexpressed in CHO cells expressing WT H 1 receptors. CHO cells were stimulated with or without 100 µM histamine for 10 min, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, YFP-arrestin1/2, and β-actin are presented in the left panels. An arrowhead indicates phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control without histamine in the right graphs. Values represent the mean ± SE of four independent experiments. ** p < 0.01, *** p < 0.001 vs. control; ## p < 0.01 vs. vehicle with histamine (one-way ANOVA followed by Tukey’s test). ( B , C ) Time-dependent effects of histamine-induced CREB phosphorylation in cells with β-arrestin1 ( B ) or β-arrestin2 ( C ) overexpression. Cells with normal expression (NE) or overexpression (OE) of β-arrestin1/2 were stimulated with histamine for the indicated times, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are depicted in the upper panels. Arrowheads indicate phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control (β-arrestin1/2 normal expression) without histamine in the bottom graphs. Values represent the mean ± SE of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (β-arrestin1/2 normal expression) without histamine. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. β-arrestin1/2 normal expression with histamine (one-way ANOVA followed by Tukey’s test). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

Article Snippet: The membranes were then incubated with primary antibodies against CREB (1:4000; #4820, Cell Signaling Technology, Danvers, MA, USA), phosphorylated CREB (1:4000; #9198, Cell Signaling Technology), β-arrestin1 (1:4000; ab32096, Abcam), and β-arrestin2 (1:4000; ab54790, Abcam) for 1 h and then reacted with peroxidase-conjugated secondary antibody (1:4000; #12-348 or #12-349, MilliporeSigma) for 30 min. After detecting CREB or arrestins, antibodies were stripped by incubating the membranes with WB Stripping Solution (Nacalai Tesque) for 5 min, and the membranes were washed with Tris-buffered saline with Tween 20 (Nacalai Tesque).

Techniques: Over Expression, Phospho-proteomics, Expressing, Western Blot, Control, Binding Assay

Journal: Pharmaceuticals

Article Title: Histamine H 1 Receptor-Mediated CREB Phosphorylation via G q Protein Signaling and Arrestin Modulation

doi: 10.3390/ph19020227

Figure Lengend Snippet: Effects of arrestin knockdown on CREB phosphorylation. ( A ) Knockdown of β-arrestin1 or β-arrestin2 was performed via siRNA treatment in CHO cells expressing WT H 1 receptors. Forty-eight hours after siRNA treatment, CHO cells were stimulated with or without 100 µM histamine for 10 min, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, β-arrestin1 , β-arrestin2 , and β-actin are depicted in the left panels. An arrowhead indicates phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control siRNA without histamine in the upper graphs. The expression levels of β-arrestin1 or β-arrestin2 are indicated in the lower graphs. Values represent the mean ± SE of four independent experiments. * p < 0.05, *** p < 0.001 vs. vehicle without histamine (one-way ANOVA followed by Tukey’s test). ( B ,C) Time-dependent effects of histamine-induced CREB phosphorylation after β-arrestin1 ( B ) or β-arrestin 2 ( C ) knockdown. Forty-eight hours after β-arrestin1 or β-arrestin 2 siRNA treatment, CHO cells were stimulated with histamine for the indicated times, and protein extracts were subjected to immunoblot analyses. Representative immunoblot images of phosphorylated CREB (phospho-CREB), total CREB, and β-actin are depicted in the upper panels. Arrowheads show phospho-CREB (43 kDa). Histamine-induced changes in the ratios of phosphorylated CREB to total CREB (phospho-CREB/total CREB) and total CREB to β-actin (total CREB/β-actin) are presented as percentages of the control siRNA without histamine in the lower graphs. Values represent the mean ± SE of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control siRNA without histamine # p < 0.05, ## p < 0.01 vs. control siRNA treatment with histamine (one-way ANOVA followed by Tukey’s test). CHO, Chinese hamster ovary; CREB, cAMP response element-binding protein; WT, wild-type.

Article Snippet: The membranes were then incubated with primary antibodies against CREB (1:4000; #4820, Cell Signaling Technology, Danvers, MA, USA), phosphorylated CREB (1:4000; #9198, Cell Signaling Technology), β-arrestin1 (1:4000; ab32096, Abcam), and β-arrestin2 (1:4000; ab54790, Abcam) for 1 h and then reacted with peroxidase-conjugated secondary antibody (1:4000; #12-348 or #12-349, MilliporeSigma) for 30 min. After detecting CREB or arrestins, antibodies were stripped by incubating the membranes with WB Stripping Solution (Nacalai Tesque) for 5 min, and the membranes were washed with Tris-buffered saline with Tween 20 (Nacalai Tesque).

Techniques: Knockdown, Phospho-proteomics, Expressing, Western Blot, Control, Binding Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: CGRP-dependent molecular signaling drives bone marrow stem cell osteogenesis in distraction osteogenesis

doi: 10.3389/fbioe.2025.1641476

Figure Lengend Snippet: CGRP activates the cAMP/PKA signaling pathway in BMSCs: (A) ELISA quantification of intracellular cAMP levels 30 min post-treatment. CGRP rapidly increased cAMP concentration, blocked by CGRP8-37 and H89. Data represent mean ± SD (n = 6). (B-D) Western blot analysis of PKA and CREB phosphorylation at 30 min. Representative blots and quantitative analysis of p-PKA/PKA and p-CREB/CREB ratios. Data represent mean ± SD. *P < 0.05.

Article Snippet: Primary antibody working solutions were then applied, including rabbit polyclonal anti-Runx2 (1:200; Bioss, bs-1134R, Woburn, MA, United States), rabbit polyclonal anti-Osterix (Osx) (1:100; ServiceBio, GB111900 , Wuhan, China), mouse monoclonal anti-osteocalcin (OCN) (1:150; ServiceBio, GB115684 ), rabbit polyclonal anti-osteopontin (OPN) (1:200; ServiceBio, GB11500), rabbit monoclonal anti-phosphorylated PKA (p-PKA Thr197) (1:100; Affinity Biosciences, #AF7246), and rabbit monoclonal anti-phosphorylated CREB (p-CREB Ser133) (1:100; Affinity Biosciences, #AF3189, Cincinnati, OH, United States), with sections placed in humid chambers at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Phospho-proteomics

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: CGRP-dependent molecular signaling drives bone marrow stem cell osteogenesis in distraction osteogenesis

doi: 10.3389/fbioe.2025.1641476

Figure Lengend Snippet: CGRP upregulates osteogenic gene and protein expression via the cAMP/PKA/CREB pathway: (A) qRT-PCR analysis of osteogenic transcription factors (Runx2 and Osterix) and marker genes (OPN and OCN), along with cell cycle gene (CyclinD1), after 7 days of osteogenic induction. Data normalized to GAPDH and expressed relative to control. (B, C) Western blot analysis and quantification of osteogenic proteins and CyclinD1 expression. Data represent mean ± SD. *P < 0.05.

Article Snippet: Primary antibody working solutions were then applied, including rabbit polyclonal anti-Runx2 (1:200; Bioss, bs-1134R, Woburn, MA, United States), rabbit polyclonal anti-Osterix (Osx) (1:100; ServiceBio, GB111900 , Wuhan, China), mouse monoclonal anti-osteocalcin (OCN) (1:150; ServiceBio, GB115684 ), rabbit polyclonal anti-osteopontin (OPN) (1:200; ServiceBio, GB11500), rabbit monoclonal anti-phosphorylated PKA (p-PKA Thr197) (1:100; Affinity Biosciences, #AF7246), and rabbit monoclonal anti-phosphorylated CREB (p-CREB Ser133) (1:100; Affinity Biosciences, #AF3189, Cincinnati, OH, United States), with sections placed in humid chambers at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Marker, Control, Western Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: CGRP-dependent molecular signaling drives bone marrow stem cell osteogenesis in distraction osteogenesis

doi: 10.3389/fbioe.2025.1641476

Figure Lengend Snippet: Immunohistochemical analysis confirms activation of osteogenicmarkers and the signaling pathway: (A) representative immunohistochemical staining and quantitative analysis of osteogenic transcription factors (Runx2 and Osterix), bone matrix proteins (OPN and OCN), and pathway molecules (p-PKA and p-CREB) in distraction gap tissues at 6 weeks of consolidation. Brown staining indicates positive expression. (B) CGRP treatment significantly enhanced all markers (scale bars: 50 μm). Data represent mean ± SD. *P < 0.05.

Article Snippet: Primary antibody working solutions were then applied, including rabbit polyclonal anti-Runx2 (1:200; Bioss, bs-1134R, Woburn, MA, United States), rabbit polyclonal anti-Osterix (Osx) (1:100; ServiceBio, GB111900 , Wuhan, China), mouse monoclonal anti-osteocalcin (OCN) (1:150; ServiceBio, GB115684 ), rabbit polyclonal anti-osteopontin (OPN) (1:200; ServiceBio, GB11500), rabbit monoclonal anti-phosphorylated PKA (p-PKA Thr197) (1:100; Affinity Biosciences, #AF7246), and rabbit monoclonal anti-phosphorylated CREB (p-CREB Ser133) (1:100; Affinity Biosciences, #AF3189, Cincinnati, OH, United States), with sections placed in humid chambers at 4 °C overnight.

Techniques: Immunohistochemical staining, Activation Assay, Staining, Expressing

Journal: Metabolites

Article Title: miR-221-3p Exacerbates Obesity-Induced Insulin Resistance by Targeting SOCS1 in Adipocytes

doi: 10.3390/metabo15090572

Figure Lengend Snippet: Transfection of miR-221-3p into cultured 3T3-L1 cells exacerbates insulin resistance in vitro. Representative Western blotting and summarized data of IRS-1 ( A ), PI3K ( B ), AKT ( C ), and GLUT4 ( D ) in the 3T3-L1 cells (N = 3 independent experiments, n = 3 technical replicates per experiment). Statistical comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. Values are means ± SD, * p < 0.05, ** p < 0.01 vs. control group. # p < 0.05, ## p < 0.01 vs. PA group. & p < 0.05, && p < 0.01 vs. mimic NC group.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies: SOCS1 (1:500, #3950), insulin receptor substrate 1 (IRS-1) (1:1000, #2382), phosphorylated IRS-1 (p-IRS-1, Ser 302, 1:1000, #2384), PI3K (p85) (1:5000, #4292), phosphorylated PI3K (p-PI3K, Tyr 458, 1:1000, #4228), AKT (1:2000, #9272), phosphorylated AKT (p-AKT, Ser 473, 1:1000, #4060), and GLUT4 (1:1000, #2213), all sourced from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Transfection, Cell Culture, In Vitro, Western Blot, Control

Journal: Metabolites

Article Title: miR-221-3p Exacerbates Obesity-Induced Insulin Resistance by Targeting SOCS1 in Adipocytes

doi: 10.3390/metabo15090572

Figure Lengend Snippet: miR-221-3p inhibition protects glucose metabolism and insulin resistance in 3T3-L1 by binding to SOCS1. ( A ) Western blotting (WB) analysis of proteins. Representative WB of p-IRS1/IRS1 ( B ), p-PI3K/PI3K ( C ), p-AKT/AKT ( D ), and GLUT4 ( E ) in 3T3-L1 cells (N = 3 independent experiments, n = 6 technical replicates per experiment). Values are means ± SD, statistical comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01 vs. control group. ## p < 0.01 vs. PA group, inhibitor NC group. $ p < 0.05, $$ p < 0.01 vs. miR-221-3p inhibitor group.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies: SOCS1 (1:500, #3950), insulin receptor substrate 1 (IRS-1) (1:1000, #2382), phosphorylated IRS-1 (p-IRS-1, Ser 302, 1:1000, #2384), PI3K (p85) (1:5000, #4292), phosphorylated PI3K (p-PI3K, Tyr 458, 1:1000, #4228), AKT (1:2000, #9272), phosphorylated AKT (p-AKT, Ser 473, 1:1000, #4060), and GLUT4 (1:1000, #2213), all sourced from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Inhibition, Binding Assay, Western Blot, Control

Journal: Pharmaceutics

Article Title: Anti-Neuroinflammation Effect of Standardized Ethanol Extract of Leaves of Perilla frutescens var. acuta on Aβ-Induced Alzheimer’s Disease-like Mouse Model

doi: 10.3390/pharmaceutics17081045

Figure Lengend Snippet: The effect of PE on LPS-induced changes in CREB phosphorylation and BDNF expression in BV2 microglial cells. BV2 microglial cells were pretreated with PE for 2 h, followed by stimulation with LPS (50 ng/mL). Protein expression was analyzed by Western blot. ( A ) pCREB expression normalized to total CREB. ( B ) mature BDNF expression normalized to β-actin. Data are presented as means ± SDs. Statistical analysis was performed using one-way ANOVA followed by the Newman–Keuls post hoc test. # p < 0.05, ### p < 0.001 vs. normal (NOR) group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. LPS-treated control (CON) group.

Article Snippet: Rabbit polyclonal phosphorylated SAPK/JNK (pJNK), rabbit polyclonal SAPK/JNK, rabbit polyclonal p38 MAPK, rabbit polyclonal cleaved caspase-3, rabbit polyclonal caspase-3, rabbit monoclonal phosphorylated CREB (pCREB), rabbit monoclonal CREB antibodies, and RIPA buffer were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Expressing, Western Blot, Control

Journal: Pharmaceutics

Article Title: Anti-Neuroinflammation Effect of Standardized Ethanol Extract of Leaves of Perilla frutescens var. acuta on Aβ-Induced Alzheimer’s Disease-like Mouse Model

doi: 10.3390/pharmaceutics17081045

Figure Lengend Snippet: Effect of PE on Aβ-induced changes in pCREB/BDNF signaling. Western blot analysis was performed on hippocampal tissue from Aβ-injected mice to assess the effect of PE on CREB/BDNF signaling. Mice were administered PE (500 mg/kg, p.o.) once daily for 7 d after Aβ injection. ( A ) pCREB normalized to total CREB. ( B ) BDNF expression normalized to GAPDH. Data are presented as means ± SDs. Statistical analysis was performed using one-way ANOVA followed by the Newman–Keuls post hoc test. # p < 0.05 vs. normal (NOR) group; * p < 0.05 vs. Aβ-injected control (CON) group.

Article Snippet: Rabbit polyclonal phosphorylated SAPK/JNK (pJNK), rabbit polyclonal SAPK/JNK, rabbit polyclonal p38 MAPK, rabbit polyclonal cleaved caspase-3, rabbit polyclonal caspase-3, rabbit monoclonal phosphorylated CREB (pCREB), rabbit monoclonal CREB antibodies, and RIPA buffer were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Injection, Expressing, Control

Journal: Cell reports

Article Title: Cholesterol metabolism regulated by CAMKK2-CREB signaling promotes castration-resistant prostate cancer

doi: 10.1016/j.celrep.2025.115792

Figure Lengend Snippet: (A) GSEA analysis of cAMP pathway and CREB motif signatures along the stat score rank of transcripts expressed in CAMKK2 KO compared with Cas9 control (WT) C4-2 cells. (B) Immunoblot and densitometry (left) and densitometry Pearson correlation (right) analysis of AR, CAMMK2, and p -CREB (Ser133) levels in 11 prostate cancer PDX models. (C) Immunoblot analysis of LNCaP and LNCaP-derived CRPC cells (C4-2 and C4-2B). (D) Immunohistochemical (IHC) staining of p -CREB (representative in dissected prostate lobes: left, 100×; quantification: right) in 40-week-old Pb-Cre4 +/+ ; Pten f/f ( n = 3) and Pb-Cre Tg/+ ; Pten f/f ( n = 3) mice. Scale bar, 100 μM. (E) IHC staining of p -CREB (representative prostate staining: left, 100×; quantification: right) in 30-week-old WT ( n = 5) and TRAMP ( n = 5) mice. Scale bar, 100 μM.

Article Snippet: Rabbit monoclonal anti-phosphorylated CREB (Ser133) (87G3) , Cell Signaling Technology , 9198 RRID:AB_2561044.

Techniques: Control, Western Blot, Derivative Assay, Immunohistochemical staining, Immunohistochemistry, Staining