Journal: Stem Cells International

Article Title: Matricellular Protein SMOC2 Potentiates BMP9-Induced Osteogenic Differentiation in Mesenchymal Stem Cells through the Enhancement of FAK/PI3K/AKT Signaling

doi: 10.1155/2023/5915988

Figure Lengend Snippet: SMOC2 potentiates BMP9-induced ectopic bone formation in MEF implantation in vivo, while knockdown of FAK inhibits bone formation. (a) Macrographic images of ectopic bone mass. MEFs were implanted subcutaneously after infection with the designed adenoviruses. Ectopic osseous masses were retrieved at 4 weeks. Representative images are shown. (b) Masson's trichrome staining results showed the effect of SMOC2 and FAK knockdown on BMP9-induced ectopic bone masses in MEFs (scale bar is 50 μ m for the panel). (c) Hematoxylin-eosin (H&E) staining results showed that the osteogenesis ability was affected by SMOC2 and/or FAK knockdown in MEFs (scale bar is 200 μ m for upper panel and 50 μ m for lower panel). (d) Immunohistochemical staining results showed the expression relationship between p-FAK and p-AKT1/2/3. Ectopic osseous masses were retrieved at 4 weeks. The expression of p-FAK and p-AKT1/2/3 was assessed by immunohistochemical staining analysis. Representative images are shown (scale bar is 50 μ m for the panel).

Article Snippet: Primary antibodies against RUNX2 (12556S), p-AKT1/2/3 (4060S), and AKT1/2/3 (9272) were purchased from Cell Signaling Technology (Shanghai, China).

Techniques: In Vivo, Infection, Staining, Immunohistochemical staining, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Pharmacological inhibition of the SKP2/p300 signaling axis restricts castration-resistant prostate cancer

doi: 10.1016/j.neo.2023.100890

Figure Lengend Snippet: SKP2 acetylation is regulated by androgen and estrogen in prostate and breast cancer cells, respectively. A . IB analysis of SKP2-IP and WCL derived from LNCaP cells. Before harvesting, cells were cultured under androgen-depleted condition and stimulated with and without insulin (Ins) or dihydrotestosterone (DHT) for 2 hours. B . IB analysis of SKP2-IP and WCL derived from castration-resistant C4-2 and ABL cell lines. Before harvesting, cells were cultured in hormone-free medium for 48 hours and then stimulated with and without insulin or DHT for 2 hours. C . IB analysis of SKP2-IP and WCL derived from ZR75-1 breast cancer cells starved in hormone-free medium and stimulated with insulin or DHT for 2 hours. pS473-AKT1 is shown as a readout of insulin stimulation. D . IB analysis of SKP2-IP and WCL derived from T47D breast cancer cells infected with the indicated lentiviral shRNAs. Before harvesting, cells were cultured in hormone-free medium for 48 hours and then treated with insulin or DHT for 2 hours.

Article Snippet: Anti-Acetylated Lysine antibody (9441), anti-SKP2 antibody (2652), anti-p300 antibody (86377), anti-p27 antibody (3686), anti-Lamin A/C antibody (4777), anti-AKT1 antibody (2938), anti-phospho-AKT1 (Ser473) antibody (9018), anti-E-cadherin antibody (4065), and anti-SIRT3 antibody (2627) were purchased from Cell Signaling Technology.

Techniques: Derivative Assay, Cell Culture, Infection

Journal: Frontiers in Immunology

Article Title: Whole genome methylation combined with RNA-seq reveals the protective effects of Gualou-Xiebai herb pair in foam cells through DNA methylation mediated PI3K-AKT signaling pathway

doi: 10.3389/fimmu.2023.1054014

Figure Lengend Snippet: Effects of GXHP on protein expressions of PI3K-AKT in foam cells. (A) RAW 264.7 cells were treated with ox-LDL or GXHP (0, 0.2, 0.6, and 1.8 g/L) for 48 h, and western blotting was performed to determine the protein levels of PI3K, p-PI3K, AKT1, and p-AKT1. Gray values of (B) p-PI3K/PI3K and (C) p-AKT/AKT are shown. Data are presented as the mean ± SD of three independent experiments. ** P < 0.01 compared with the control group; # P < 0.05 compared with the model group; ## P < 0.01 compared with the model group.

Article Snippet: After blocking, the PVDF membrane was incubated with specific primary antibodies (Cell Signaling Technology, Danvers, MA, USA), such as PI3 kinase p85 antibody (dilution ratio was 1:500), phospho-PI3 kinase p85 antibody (dilution ratio was 1:1,000), AKT1 antibody (dilution ratio was 1:1500), and phospho-AKT1 antibody (dilution ratio was 1:500), overnight at 4°C.

Techniques: Western Blot

Journal: Frontiers in Aging Neuroscience

Article Title: Identification and evaluation of midbrain specific longevity-related genes in exceptionally long-lived but healthy mice

doi: 10.3389/fnagi.2022.1030807

Figure Lengend Snippet: Upregulation of neuroprotective NRF1, parkin, and phosphorylated Akt in the midbrain of exceptionally long-lived healthy mice. (A) Representative western blots of NRF1, pAkt1, Akt1, pAkt2, and Akt2 in the midbrain of mice at 8 and 34 months of age using the indicated antibodies. β-actin was used as an internal loading control. (B) Quantification of the relative expression levels of the indicated proteins in the midbrain of mice aged 8 and 34 months normalized to β-actin ( n = 4 mice for 8 months; n = 6 mice for 34 months). (C) Representative western blots of PARIS, Mfn2, PINK1, parkin, and AIMP2 in the midbrain of mice aged 8 and 34 months using the indicated antibodies. β-actin was used as an internal loading control. (D) Quantification of the relative expression levels of the indicated proteins in the midbrain of 8-month-old and 34-month-old mice normalized to β-actin ( n = 4 mice for 8 months; n = 6 mice for 34 months). (E) Representative western blots of GFAP and TH in the midbrain of mice aged 8 and 34 months using the indicated antibodies. β-actin was used as an internal loading control. (F) Quantification of the relative expression levels of the indicated proteins in the midbrain of mice aged 8 and 34 months normalized to β-actin ( n = 4 mice for 8 months; n = 6 mice for 34 months). Quantified data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01, and *** p < 0.001, unpaired two-tailed Student’s t -test.

Article Snippet: The following primary antibodies were used: rabbit antibodies for TH (#NB300-109, 1:5,000 for WB, 1:1,000 for IHC, Novus Biologicals), rabbit antibodies for NRF1 (#ab34682, 1:5,000, Abcam), rabbit antibodies for GFAP (#ab7260, 1:5,000 for WB, 1:2,500 for IHC, Abcam), rabbit antibody for AIMP2 (#10424-1-AP, 1:5,000, Proteintech), rabbit antibody for PARIS (#24543-1-AP, 1:5,000, Proteintech), rabbit antibody for Mfn2 (#9482S, 1:5,000, Cell Signaling Technology), rabbit antibody for PINK1 (#BC100-494, 1:3,000, Novus Biologicals), rabbit antibody for phospho-Akt1(S473) (#9018S, 1:5,000, Cell Signaling Technology), rabbit antibody for phospho-Akt2 (S474) (#8599S, 1:5,000, Cell Signaling Technology), rabbit antibody for IBA1 (#019-19,741, 1:500, Wako), mouse antibody for ubiquitin (#sc-8017, 1:1,000, Santa Cruz), mouse antibody for p21 (#sc-6246, 1:500, Santa Cruz), mouse antibody for parkin (#4211S, 1:5,000, Cell Signaling Technology), mouse antibody for Akt1 (#2967S, 1:5,000, Cell Signaling Technology), mouse antibody for Akt2 (#5239S, 1:5,000, Cell Signaling Technology), horse radish peroxidase (HRP)-conjugated mouse antibody for HA (#2999S, 1:5,000, Cell Signaling Technology), and HRP-conjugated mouse antibody for FLAG (#8592, 1:5,000, Sigma).

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: Frontiers in Aging Neuroscience

Article Title: Identification and evaluation of midbrain specific longevity-related genes in exceptionally long-lived but healthy mice

doi: 10.3389/fnagi.2022.1030807

Figure Lengend Snippet: Regulation of longevity-related genes in the midbrain of aged mouse brains. (A) Representative western blots of PARIS, parkin and AIMP2 in the midbrain of mice aged 8 and 24 months using the indicated antibodies. β-actin was used as an internal loading control. (B) Quantification of the relative expression levels of the indicated proteins in the midbrain of mice aged 8 and 24 months normalized to β-actin ( n = 4 mice per group). (C) Representative western blots of NRF1, pAkt1 and Akt1 in the midbrain of mice aged 8 and 24 months using the indicated antibodies. β-actin was used as an internal loading control. (D) Quantification of the relative expression levels of the indicated proteins in the midbrain of 8-month-old and 24-month-old mice normalized to β-actin ( n = 4 mice per group). Quantified data are expressed as mean ± SEM * p < 0.05 and ** p < 0.01, unpaired two-tailed Student’s t -test; n.s., non-significant.

Article Snippet: The following primary antibodies were used: rabbit antibodies for TH (#NB300-109, 1:5,000 for WB, 1:1,000 for IHC, Novus Biologicals), rabbit antibodies for NRF1 (#ab34682, 1:5,000, Abcam), rabbit antibodies for GFAP (#ab7260, 1:5,000 for WB, 1:2,500 for IHC, Abcam), rabbit antibody for AIMP2 (#10424-1-AP, 1:5,000, Proteintech), rabbit antibody for PARIS (#24543-1-AP, 1:5,000, Proteintech), rabbit antibody for Mfn2 (#9482S, 1:5,000, Cell Signaling Technology), rabbit antibody for PINK1 (#BC100-494, 1:3,000, Novus Biologicals), rabbit antibody for phospho-Akt1(S473) (#9018S, 1:5,000, Cell Signaling Technology), rabbit antibody for phospho-Akt2 (S474) (#8599S, 1:5,000, Cell Signaling Technology), rabbit antibody for IBA1 (#019-19,741, 1:500, Wako), mouse antibody for ubiquitin (#sc-8017, 1:1,000, Santa Cruz), mouse antibody for p21 (#sc-6246, 1:500, Santa Cruz), mouse antibody for parkin (#4211S, 1:5,000, Cell Signaling Technology), mouse antibody for Akt1 (#2967S, 1:5,000, Cell Signaling Technology), mouse antibody for Akt2 (#5239S, 1:5,000, Cell Signaling Technology), horse radish peroxidase (HRP)-conjugated mouse antibody for HA (#2999S, 1:5,000, Cell Signaling Technology), and HRP-conjugated mouse antibody for FLAG (#8592, 1:5,000, Sigma).

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: Frontiers in Aging Neuroscience

Article Title: Identification and evaluation of midbrain specific longevity-related genes in exceptionally long-lived but healthy mice

doi: 10.3389/fnagi.2022.1030807

Figure Lengend Snippet: AIMP2 toxicity is inhibited by longevity-related and neuroprotective Akt1, NRF1, and parkin expression. (A) Confirmation of protein expression in SH-SY5Y cells transiently transfected (48 h) with Flag-tagged NRF1, parkin, AIMP2, HA-tagged Akt1, and Akt2 determined by a western blot using the indicated antibodies. (B) Cell viability assessment for SH-SY5Y cells expressing AIMP2 alone or in combination with NRF1, Akt1, Akt2, and parkin (48 h) determined by a trypan blue exclusion assay ( n = 6 per group). (C) Functional assessment of mitochondrial membrane potential for SH-SY5Y cells transiently transfected with the indicated constructs (48 h) determined by fluorescence reading using a JC-1 dye. Red and green fluorescences reflect healthy and damaged mitochondria, respectively. Scale bar = 10 μm. (D) Relative mitochondrial red/green fluorescence ratio in SH-SY5Y cells transiently transfected with the indicated constructs (48 h) and stained with a JC-1 dye ( n = 13 images from 3 experiments per group). (E) Representative fluorescence images, paths of cell body, and neurites of the differentiated (+NGF, 42 h) PC12 cells expressing GFP-tagged AIMP2 in combination with constitutively active Akt1, NRF1, or parkin (48 h). Scale bar, 10 μm. (F) Quantification of the relative average lengths of the neurites from differentiated PC12 cells expressing the indicated combination of proteins ( n = 30 cells per group in 5 experiments per group). Scale bar = 10 μm. Quantified data are expressed as mean ± SEM *** p < 0.001, ANOVA test followed by Tukey’s HSD post hoc analysis.

Article Snippet: The following primary antibodies were used: rabbit antibodies for TH (#NB300-109, 1:5,000 for WB, 1:1,000 for IHC, Novus Biologicals), rabbit antibodies for NRF1 (#ab34682, 1:5,000, Abcam), rabbit antibodies for GFAP (#ab7260, 1:5,000 for WB, 1:2,500 for IHC, Abcam), rabbit antibody for AIMP2 (#10424-1-AP, 1:5,000, Proteintech), rabbit antibody for PARIS (#24543-1-AP, 1:5,000, Proteintech), rabbit antibody for Mfn2 (#9482S, 1:5,000, Cell Signaling Technology), rabbit antibody for PINK1 (#BC100-494, 1:3,000, Novus Biologicals), rabbit antibody for phospho-Akt1(S473) (#9018S, 1:5,000, Cell Signaling Technology), rabbit antibody for phospho-Akt2 (S474) (#8599S, 1:5,000, Cell Signaling Technology), rabbit antibody for IBA1 (#019-19,741, 1:500, Wako), mouse antibody for ubiquitin (#sc-8017, 1:1,000, Santa Cruz), mouse antibody for p21 (#sc-6246, 1:500, Santa Cruz), mouse antibody for parkin (#4211S, 1:5,000, Cell Signaling Technology), mouse antibody for Akt1 (#2967S, 1:5,000, Cell Signaling Technology), mouse antibody for Akt2 (#5239S, 1:5,000, Cell Signaling Technology), horse radish peroxidase (HRP)-conjugated mouse antibody for HA (#2999S, 1:5,000, Cell Signaling Technology), and HRP-conjugated mouse antibody for FLAG (#8592, 1:5,000, Sigma).

Techniques: Expressing, Transfection, Western Blot, Trypan Blue Exclusion Assay, Functional Assay, Construct, Fluorescence, Staining

Journal: Frontiers in Psychiatry

Article Title: Profiling prefrontal cortex protein expression in rats exhibiting an incubation of cocaine craving following short-access self-administration procedures

doi: 10.3389/fpsyt.2022.1031585

Figure Lengend Snippet: Changes in Akt1 and CaMKII expression and phosphorylation in the PL. While we did not detect any time-dependent or group differences in the PL expression of total Akt1 (A) , both 2-h and Mixed rats exhibited a time-dependent increase total p(Ser473)-Akt1 expression (B) and the 2-h rats exhibited a time-dependent increase in the relative expression of p(Ser473)-Akt1 (C) . (D) We detected no group or time-dependent changes in total CaMKII expression within the PL. However, the Mixed group demonstrated a significant time-dependent increase in both total (E) and relative (F) p(Thr286)-CaMKII expression. Data are normalized to the average protein densities of the Sham controls tested on WD3 and represented as means ± SEMs of the number of rats indicated. + p < 0.05, compared to WD3.

Article Snippet: The following mouse primary antibodies were also employed: Homer1b/c (1:1,000 dilution; Santa Cruz Biotechnology; sc-25271), mGlu1 (1:1,000 dilution; BD Biosciences; 610965), p(Ser473)-Akt1 (1:1,000 dilution; Cell Signaling Technology; 4051), GluN2b (1:500 dilution; Invitrogen; MA1-2014), GluA2 (1:500 dilution; Synaptic Systems; 182 111), and CaMKII (1:500 dilution; Millipore; 05-532).

Techniques: Expressing

Journal: Frontiers in Psychiatry

Article Title: Profiling prefrontal cortex protein expression in rats exhibiting an incubation of cocaine craving following short-access self-administration procedures

doi: 10.3389/fpsyt.2022.1031585

Figure Lengend Snippet: Summary of the negative results from the immunoblotting study of the IL from Sham, 2-h- and Mixed-Access rats.

Article Snippet: The following mouse primary antibodies were also employed: Homer1b/c (1:1,000 dilution; Santa Cruz Biotechnology; sc-25271), mGlu1 (1:1,000 dilution; BD Biosciences; 610965), p(Ser473)-Akt1 (1:1,000 dilution; Cell Signaling Technology; 4051), GluN2b (1:500 dilution; Invitrogen; MA1-2014), GluA2 (1:500 dilution; Synaptic Systems; 182 111), and CaMKII (1:500 dilution; Millipore; 05-532).

Techniques: Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Multifunctional Effects of Mangosteen Pericarp on Cognition in C57BL/6J and Triple Transgenic Alzheimer's Mice

doi: 10.1155/2014/813672

Figure Lengend Snippet: List of the primary antibodies used in this study.

Article Snippet: pAkt 1/2 , Rabbit , Cell Signaling , 1 : 1,000 , — , Akt phosphorylated at Thr 450.

Techniques: Binding Assay