p pi3k tyr607  (Boster Bio)

Journal: Biomolecules and Biomedicine

Article Title: Therapeutic potential of simvastatin in ALS: Enhanced axonal integrity and motor neuron survival through Apoa4 and Alb modulation

doi: 10.17305/bb.2024.11218

Figure Lengend Snippet: PCR primers and conditions

Article Snippet: Primary antibodies: C4a (1:3000, 22233-1-AP, Proteintech, Wuhan, China); C5 (1:600, A8104, Abclonal, Wuhan, China); C3 (1:10,000, 21337-1-AP, Proteintech); PI3K (1:1000, ab302958, Abcam, UK); p-PI3K p85 alpha (Tyr607) (1:1000, AF3241, Affinity, Jiangsu, China); Alb (1:10,000, 16475-1-AP, Proteintech); p-AKT (Ser473) (1:10,000, 66444-1-Ig, Proteintech); AKT (1:5000, 60203-2-Ig, Proteintech); PPARγ (1:5000, 16643-1-AP, Proteintech); APOA4 (1:8000, 17996-1-AP, Proteintech); C1qB (1:1000, A5339, Abclonal, Wuhan, China); CRP (1:2000, 66250-1-Ig, Proteintech); and GAPDH (1:10,000, 60004-1-Ig, Proteintech).

Techniques:

Journal: Biomolecules and Biomedicine

Article Title: Therapeutic potential of simvastatin in ALS: Enhanced axonal integrity and motor neuron survival through Apoa4 and Alb modulation

doi: 10.17305/bb.2024.11218

Figure Lengend Snippet: Expression of mRNA and proteins. qRT-PCR results presented the expression levels of PI3K (A), AKT (B), C5 (C), PPARγ (D), C4a (E), C3 (F), C1qB (G), and CRP (H) mRNA in different groups; the expression levels of PI3K, p-PI3K, p-AKT, AKT, PPARγ, Apoa4 (I), and C4a, C5, C3, Alb, C1qB, and CRP (J) in different groups were detected. The levels of p-PI3K, p-AKT, PPARγ, Apoa4, C5, and Alb decreased, while C4a, C3, C1qB, and CRP increased in the SOD1G93A + PBS group compared with the WT group; * P < 0.05, ** P < 0.01, vs WT group; # P < 0.05, ## P < 0.01, vs SOD1 G93A + PBS group. qRT-PCR: Quantitative real-time polymerase chain reaction; SOD1: Superoxide dismutase 1; WT: Wilt-type; PPAR: Peroxisome proliferator-activated receptor.

Article Snippet: Primary antibodies: C4a (1:3000, 22233-1-AP, Proteintech, Wuhan, China); C5 (1:600, A8104, Abclonal, Wuhan, China); C3 (1:10,000, 21337-1-AP, Proteintech); PI3K (1:1000, ab302958, Abcam, UK); p-PI3K p85 alpha (Tyr607) (1:1000, AF3241, Affinity, Jiangsu, China); Alb (1:10,000, 16475-1-AP, Proteintech); p-AKT (Ser473) (1:10,000, 66444-1-Ig, Proteintech); AKT (1:5000, 60203-2-Ig, Proteintech); PPARγ (1:5000, 16643-1-AP, Proteintech); APOA4 (1:8000, 17996-1-AP, Proteintech); C1qB (1:1000, A5339, Abclonal, Wuhan, China); CRP (1:2000, 66250-1-Ig, Proteintech); and GAPDH (1:10,000, 60004-1-Ig, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: FASEB BioAdvances

Article Title: NCAPD3 ‐mediated AKT activation regulates prostate cancer progression

doi: 10.1096/fba.2024-00073

Figure Lengend Snippet: NCAPD3 upregulated the phosphorylation level of AKT (T308) via JAK2/PI3K axis. (A) Protein levels of PI3K p110β, PI3K p85, AKT, PTEN, p‐PI3K p85, and p‐AKT (T308) assayed by Western blot in NCAPD3 overexpression or knockdown PCa cells. (B) Protein expressions of NCAPD3, AKT, p‐AKT (T308), FOXO1, and p‐FOXO1 (S256) in DU145 cells with transfection of NCAPD3 and treatment of LY294002 (20 μM). (C and D) Levels of JAK2 protein and mRNA expressions in NCAPD3‐overexpression DU145 cells and NCAPD3‐knockdown LNCaP cells. (E) Protein levels of NCAPD3, PI3K, p‐PI3K, AKT, and p‐AKT (T308) in DU145 cells with transfection of NCAPD3 and treatment of Z3 (30 μM). Values are means ± SE from n = 3 independent repetitions, * p < 0.05, ** p < 0.01, *** p < 0.001, based on Student's t ‐test.

Article Snippet: Antibodies against p‐FoxO1(Ser256) (Cat#AF3417), RICTOR (Cat#DF7530), RAPTOR (Cat#DF7527), PI3K p85α (Cat#AF6241), p‐PI3K p85α (Tyr607) (Cat#AF3241), and p‐STAT3(Y705) (Cat#AF3293) were from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Western Blot, Over Expression, Knockdown, Transfection

Journal: FASEB BioAdvances

Article Title: NCAPD3 ‐mediated AKT activation regulates prostate cancer progression

doi: 10.1096/fba.2024-00073

Figure Lengend Snippet: NCAPD3 increased the level of STAT3 and recruited more STAT3 to the promoters of EZH2 and JAK2 and then activated EZH2/NSD2/mTORC2 and JAK2/PI3K pathways, which phosphorylated AKT at Thr 308 and Ser 473. Moreover, there is a positive mutual activation between STAT3 and JAK2, further enhanced by NCAPD3 to promote PCa progression. The graph was drawn by Figdraw.

Article Snippet: Antibodies against p‐FoxO1(Ser256) (Cat#AF3417), RICTOR (Cat#DF7530), RAPTOR (Cat#DF7527), PI3K p85α (Cat#AF6241), p‐PI3K p85α (Tyr607) (Cat#AF3241), and p‐STAT3(Y705) (Cat#AF3293) were from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Activation Assay

Journal: Cancer Medicine

Article Title: Selenium‐binding protein 1 inhibits malignant progression and induces apoptosis via distinct mechanisms in non‐small cell lung cancer

doi: 10.1002/cam4.6309

Figure Lengend Snippet: Overexpression of SELENBP1 suppressed the proliferation, migration, and invasion of NSCLC cells in part via inactivating the PI3K/AKT signaling pathway in vitro. (A) Western blotting analysis of PI3K/AKT/mTOR pathway markers in A549‐SELENBP1, H1299‐SELENBP1, and control cells. The NSCLC cell lysate was collected and subjected to western blotting analysis with antibodies against members of the PI3K/AKT/mTOR pathway. Levels of β‐Actin were used as loading control. (B) The histogram was used to quantify the experimental results of western blotting in A549‐SELENBP1 and their control cells, and H1299‐SELENBP1 and their control cells. All data were presented as the mean ± SD, unpaired t ‐test, “*” p < 0.05, “**” p < 0.01, “****” p < 0.001, A549‐Control group versus A549‐SELENBP1 group and H1299‐Control group versus H1299‐SELENBP1 group. (C) Western blotting analysis of PI3K/mTOR pathway markers in the indicated cells treated with LY294002, MK‐2206, or IGF‐1 at various concentrations. (D) CCK8 was performed at 0, 24, 48 and 72 h in the indicated cells treated with LY294002 (20 μM), MK‐2206 (1 μM), or IGF‐1 (20 μg/mL; N = 4). And all data were presented as the mean ± SD. Multiple groups were compared by using one‐way ANOVA. Two groups were compared using unpaired t ‐test “**” p < 0.01. A549‐Control group versus A549‐Control‐LY294002 group, A549‐Control group versus A549‐Control‐MK2206 group, A549‐SELENBP1 group versus A549‐SELENBP1‐IGF‐1 group. H1299‐Control group versus H1299‐Control‐LY294002 group, H1299‐Control group versus H1299‐Control‐MK2206 group, and H1299‐SELENBP1 group versus H1299‐SELENBP1‐IGF‐1 group. (E) The number of migrated cells and the number of invasion cells in indicated cells were measured by Millicell assay and transwell assay, respectively (left). Scale bars, 20 μm. The histogram was used to quantify the experimental results (right), and all data were presented as the mean ± SD. Multiple groups were compared by using one‐way ANOVA. Two groups were compared using unpaired t ‐test “**” p < 0.01. “***” p < 0.005. A549‐Control group versus A549‐Control‐LY294002 group, A549‐Control group versus A549‐Control‐MK2206 group, A549‐SELENBP1 group versus A549‐SELENBP1‐IGF‐1 group. H1299‐Control group versus H1299‐Control‐LY294002 group, H1299‐Control group versus H1299‐Control‐MK2206 group, and H1299‐SELENBP1 group versus H1299‐SELENBP1‐IGF‐1 group.

Article Snippet: In IHC staining, the sections were deparaffinize, rehydrated, followed by antigen retrieval and immunostaining with different antibodies against SELENBP1 (1:200 dilution, Abcam, ab90135), SP‐C (1:200 dilution, Abcam, ab90716), Ki‐67 (1:200 dilution, Abcam, ab15580), PI3K (1:100 dilution, Abcam, ab151549), p‐PI3K (Tyr607; 1:200 dilution, Invitrogen, Catalog #PA5‐104853), AKT (1:200 dilution, CST, #4691), p‐AKT (Ser473; 1:100 dilution, CST, #4060), mTOR (1:100 dilution, CST, #2983), p‐mTOR (Ser2448; 1:200 dilution, Invitrogen, Catalog #44‐1125G), Caspase‐3 (1:200 dilution, Huabio, ET1602‐39), Cleaved‐Caspase‐3 (1:200 dilution, Affinity, AF7002), Bcl‐2 (1:200 dilution, Huabio, ET1603‐11), and Bax (1:200 dilution, Affinity, AF0120).

Techniques: Over Expression, Migration, In Vitro, Western Blot, Transwell Assay

Journal: Cancer Medicine

Article Title: Selenium‐binding protein 1 inhibits malignant progression and induces apoptosis via distinct mechanisms in non‐small cell lung cancer

doi: 10.1002/cam4.6309

Figure Lengend Snippet: Overexpression of SELENBP1 inhibiting the growth of NSCLC cells in vivo was associated with the inhibition of PI3K/AKT/mTOR pathway. (A) Establishment of stable SELENBP1 overexpressing A549 cells and control cells tumor xenograft Model. Athymic nude mice (4‐week‐old male, N = 6/point/group) were subcutaneously injected A549‐SELENBP1 cells or A549‐Control cells, respectively. The mice were sacrificed after 29 days, the retrieved tumor samples were presented. (B) Volume of Xenograft tumor was measured once every 3 days after 5 days subcutaneous injection. Data were presented as the mean ± SD, unpaired t ‐test, “*” p < 0.05, “**” p < 0.01, A549‐Control tumors group versus A549‐SELENBP1 tumors group. (C) Paraffin sections of the retrieved tumor samples were subjected to H&E staining and IHC staining with antibodies against SELENBP1, Ki‐67. Staining without primary antibody was used as negative controls. Results of H&E were observed under a bright field microscope (×200), Scale bars, 50 μm. Results of IHC were observed under a bright field microscope (×400), Scale bars, 20 μm. The histogram was used to quantify the experimental results of IHC ( N = 3). All data were presented as the mean ± SD, unpaired t ‐test, “**” p < 0.01, “****” p < 0.001, A549‐Control tumors group versus A549‐SELENBP1 tumors group. (D) Paraffin sections of the retrieved tumor samples were subjected to IHC staining with antibodies against PI3K, p‐PI3K, AKT, p‐AKT, mTOR, and p‐mTOR. Staining without primary antibody was used as negative controls. Results were observed under a bright field microscope (×400). Scale bars, 20 μm. The histogram was used to quantify the experimental results of IHC ( N = 3). And all data were presented as the mean ± SD, unpaired t ‐test, “*” p < 0.05, “**” p < 0.01, “***” p < 0.005, A549‐Control tumors group versus A549‐SELENBP1 tumors group. (E) The tumor tissues were retrieved, extracted total protein, and subjected to western blotting analysis of key components of PI3K/AKT/mTOR pathway ( N = 3/group). Levels of β‐Actin were used as loading control.

Article Snippet: In IHC staining, the sections were deparaffinize, rehydrated, followed by antigen retrieval and immunostaining with different antibodies against SELENBP1 (1:200 dilution, Abcam, ab90135), SP‐C (1:200 dilution, Abcam, ab90716), Ki‐67 (1:200 dilution, Abcam, ab15580), PI3K (1:100 dilution, Abcam, ab151549), p‐PI3K (Tyr607; 1:200 dilution, Invitrogen, Catalog #PA5‐104853), AKT (1:200 dilution, CST, #4691), p‐AKT (Ser473; 1:100 dilution, CST, #4060), mTOR (1:100 dilution, CST, #2983), p‐mTOR (Ser2448; 1:200 dilution, Invitrogen, Catalog #44‐1125G), Caspase‐3 (1:200 dilution, Huabio, ET1602‐39), Cleaved‐Caspase‐3 (1:200 dilution, Affinity, AF7002), Bcl‐2 (1:200 dilution, Huabio, ET1603‐11), and Bax (1:200 dilution, Affinity, AF0120).

Techniques: Over Expression, In Vivo, Inhibition, Injection, Staining, Immunohistochemistry, Microscopy, Western Blot

Journal: Cancer Medicine

Article Title: Selenium‐binding protein 1 inhibits malignant progression and induces apoptosis via distinct mechanisms in non‐small cell lung cancer

doi: 10.1002/cam4.6309

Figure Lengend Snippet: Molecular mechanisms for overexpression of SELENBP1 inhibiting the malignant progression and inducing the apoptosis in NSCLC cells. Overexpression of SELENBP1 decreased the expression of PI3K, p‐PI3K, AKT, p‐AKT, mTOR, p‐mTOR, and Bcl‐2, whereas increased the expression of Bax and caspase‐3. Overexpression of SELENBP1 suppressed the malignant progression of NSCLC cells by inhibiting the PI3K/AKT/mTOR signaling. Moreover, overexpression of SELENBP1 inducing the apoptosis of NSCLC cells was associated with the activation of caspase‐3‐dependent axis under nonhigh level of oxidative stress. Furthermore, overexpression of SELENBP1 facilitating the cell apoptosis under high level of oxidative stress was related to its combining with GPX1 and colocalizing in nucleus (ROS, reactive oxygen species).

Article Snippet: In IHC staining, the sections were deparaffinize, rehydrated, followed by antigen retrieval and immunostaining with different antibodies against SELENBP1 (1:200 dilution, Abcam, ab90135), SP‐C (1:200 dilution, Abcam, ab90716), Ki‐67 (1:200 dilution, Abcam, ab15580), PI3K (1:100 dilution, Abcam, ab151549), p‐PI3K (Tyr607; 1:200 dilution, Invitrogen, Catalog #PA5‐104853), AKT (1:200 dilution, CST, #4691), p‐AKT (Ser473; 1:100 dilution, CST, #4060), mTOR (1:100 dilution, CST, #2983), p‐mTOR (Ser2448; 1:200 dilution, Invitrogen, Catalog #44‐1125G), Caspase‐3 (1:200 dilution, Huabio, ET1602‐39), Cleaved‐Caspase‐3 (1:200 dilution, Affinity, AF7002), Bcl‐2 (1:200 dilution, Huabio, ET1603‐11), and Bax (1:200 dilution, Affinity, AF0120).

Techniques: Over Expression, Expressing, Activation Assay