Journal: The Journal of clinical endocrinology and metabolism

Article Title: Leptin secretion by human ejaculated spermatozoa.

doi: 10.1210/jc.2004-2233

Figure Lengend Snippet: FIG. 5. Leptin and insulin action on PI3K/Akt pathway in human ejaculated spermatozoa. Washed spermatozoa were incubated in the un- supplemented Earle’s medium for 1 h at 37 C and 5% CO2, in the absence (NC) or in the presence of 10 nM leptin (Lep) or in the presence of 3.3 nM insulin (Ins) alone or combined with 10 nM leptin (Ins Lep). Some samples were washed with the unsupplemented Earle’s medium and incubated in capacitating medium for 1 h (CAP). A, A total of 500 g sperm lysates was immunoprecipitated using anti-p85 regulatory subunit of PI3K incu- bated in the presence of 200 M phosphatidylino- sitol and 10 Ci [-32P]ATP for 30 min. The neg- ative control (Neg) was performed using a sperm lysate, where p110 catalyzing subunit of PI3K was previously removed by preincubation with the respective antibody (1 h at room temperature) and subsequently immunoprecipitated with pro- tein A/G-agarose (lane 6). PI-3,4,5-P3, phospha- tidylinositol 3,4,5-triphosphate; PI-3,5-P2, phos- phatidylinositol 3,5-diphosphate. B and C, A total of 50 g sperm lysates was used for Western blot analysis of p-AKT S473 or p-GSK3 S9 (B), leptin or insulin (C). The autoradiographs presented are representative examples of experiments that were performed at least six times with repetitive results. •, P 0.05; *, P 0.001 vs. control.

Article Snippet: Monoclonal mouse p85-regulatory subunit of phosphatidyl-inositol-kinase-3 (PI3K) antibody, goat polyclonal actin antibody, polyclonal rabbit antileptin (A-20) antibody, rabbit antiinsulin antibody, rabbit antiphosphotyrosine antibody (PY99), rabbit anti-pAkt1/Akt2/Akt3 S473 antibody, POD-coupled antirabbit and antigoat, antirabbit IgG fluorescein isothiocyanate-conjugated were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Incubation, Immunoprecipitation, Control, Western Blot

Journal: BMC Cancer

Article Title: Docosahexaenoic acid reduces sterol regulatory element binding protein-1 and fatty acid synthase expression and inhibits cell proliferation by inhibiting pAkt signaling in a human breast cancer MCF-7 cell line

doi: 10.1186/s12885-017-3936-7

Figure Lengend Snippet: Effect of DHA or E 2 on the pERK1/2/ERK1/2, pAkt/Akt, and pS6/S6 ratios and p-SREBP-1, m-SREBP-1, and FASN expression in MCF-7 cells. Cells were pretreated with BSA or 60 μM BSA-bound DHA for 48 h in DMEM containing 5% CD-FBS, then the same medium alone or 10 nM E 2 was added. Western blots were then used to measure the pERK1/2/ERK1/2, pAkt/Akt, and pS6/S6 ratios after 1 h and p-SREBP-1, m-SREBP-1, and FASN expression after 24 h ( a ). Total ERK1/2, Akt, or S6 was used as the loading control for pERK1/2 ( b ), pAkt ( c ), or pS6 ( d ), respectively, while GAPDH was used as the loading control for p-SREBP-1 ( e ), m-SREBP-1 ( f ), and FASN ( g ).The levels are expressed as a fold value compared to the BSA-treated control with no E 2 stimulation. Two-way ANOVA followed by the Bonferroni posttest was used to compare DHA and E 2 effects. The data are presented as the mean ± S.E.M for 5–6 independent experiments

Article Snippet: The membranes were immunoblotted overnight at 4 °C with primary antibodies diluted in TBST; the antibodies used were rabbit monoclonal antibody against pAkt (1:1000), S6 (1:1000), or GAPDH (1:2000), rabbit polyclonal antibodies against Akt (1:1000), pS6 (1:1000), pERK1/2 (1:1000), or ERK1/2 (1:1000) (all from Cell Signaling), rabbit polyclonal anti-FASN antibodies (1:1000), or mouse monoclonal anti-SREBP1 antibody (1:200) (both from Santa Cruz).

Techniques: Expressing, Western Blot, Control

Journal: BMC Cancer

Article Title: Docosahexaenoic acid reduces sterol regulatory element binding protein-1 and fatty acid synthase expression and inhibits cell proliferation by inhibiting pAkt signaling in a human breast cancer MCF-7 cell line

doi: 10.1186/s12885-017-3936-7

Figure Lengend Snippet: Effect of DHA or insulin on the pERK1/2/ERK1/2, pAkt/Akt, and pS6/S6 ratios and p-SREBP-1, m-SREBP-1, and FASN expression in MCF-7 cells. Cells were pretreated with BSA or 60 μM BSA-bound DHA for 48 h in DMEM containing 5% FBS, then the same medium alone or 1 μg/ml of insulin was added. Western blots were then used to measure the pERK1/2/ERK1/2, pAkt/Akt, and pS6/S6 ratios after 1 h and p-SREBP-1, m-SREBP-1, and FASN expression after 24 h ( a ). Total ERK1/2, Akt, or S6 was used as the loading control for pERK1/2 ( b ), pAkt ( c ), or pS6 ( d ), respectively, while GAPDH was used as the loading control for p-SREBP-1 ( e ), m-SREBP-1 ( f ), and FASN ( g ).The levels are expressed as a fold value compared to the BSA-treated control with no insulin stimulation. Two-way ANOVA followed by the Bonferroni posttest was used to compare DHA and insulin effects. The data are presented as the mean ± S.E.M for 5–6 independent experiments

Article Snippet: The membranes were immunoblotted overnight at 4 °C with primary antibodies diluted in TBST; the antibodies used were rabbit monoclonal antibody against pAkt (1:1000), S6 (1:1000), or GAPDH (1:2000), rabbit polyclonal antibodies against Akt (1:1000), pS6 (1:1000), pERK1/2 (1:1000), or ERK1/2 (1:1000) (all from Cell Signaling), rabbit polyclonal anti-FASN antibodies (1:1000), or mouse monoclonal anti-SREBP1 antibody (1:200) (both from Santa Cruz).

Techniques: Expressing, Western Blot, Control

Journal: BMC Cancer

Article Title: Docosahexaenoic acid reduces sterol regulatory element binding protein-1 and fatty acid synthase expression and inhibits cell proliferation by inhibiting pAkt signaling in a human breast cancer MCF-7 cell line

doi: 10.1186/s12885-017-3936-7

Figure Lengend Snippet: Effect of inhibitors and DHA on the E 2 -stimulated increase in the pAkt/Akt and pS6/S6 ratios and p-SREBP-1, m-SREBP-1, and FASN expression in MCF-7 cells. Cells were pretreated with BSA or 60 μM BSA-bound DHA for 48 h in DMEM containing 5% CD-FBS, then the same medium alone, 20 μM LY294002 (LY), or 0.5 nM rapamycin (Rap) was added for 1 h, followed by stimulation with 10 nM E 2 , then Western blot analysis was used to measure the pAkt/Akt and pS6/S6 ratios after 1 h of incubation and p-SREBP-1, m-SREBP-1, and FASN expression after 24 h ( a ). Total Akt or total S6 was used as the loading control for pAkt ( b ) or pS6 ( c ), respectively, while GAPDH was used as the loading control for p-SREBP-1 ( d ), m-SREBP-1 ( e ), and FASN ( f ).The levels are expressed as a fold value compared to control BSA-treated cells. Two-way ANOVA followed by the Bonferroni posttest was used to compare DHA and inhibitor effects. The data are presented as the mean ± S.E.M for 4–5 independent experiments

Article Snippet: The membranes were immunoblotted overnight at 4 °C with primary antibodies diluted in TBST; the antibodies used were rabbit monoclonal antibody against pAkt (1:1000), S6 (1:1000), or GAPDH (1:2000), rabbit polyclonal antibodies against Akt (1:1000), pS6 (1:1000), pERK1/2 (1:1000), or ERK1/2 (1:1000) (all from Cell Signaling), rabbit polyclonal anti-FASN antibodies (1:1000), or mouse monoclonal anti-SREBP1 antibody (1:200) (both from Santa Cruz).

Techniques: Expressing, Western Blot, Incubation, Control

Journal: BMC Cancer

Article Title: Docosahexaenoic acid reduces sterol regulatory element binding protein-1 and fatty acid synthase expression and inhibits cell proliferation by inhibiting pAkt signaling in a human breast cancer MCF-7 cell line

doi: 10.1186/s12885-017-3936-7

Figure Lengend Snippet: Effect of inhibitors and DHA on the insulin-stimulated increase in the pAkt/Akt and pS6/S6 ratios and p-SREBP-1, m-SREBP-1, and FASN expression in MCF-7 cells. Cells were pretreated with BSA or 60 μM BSA-bound DHA for 48 h in DMEM containing 5% FBS, then the same medium, 20 μM LY294002 (LY), or 0.5 nM rapamycin (Rap) was added for 1 h following by stimulation with 1 μg/ml of insulin, then Western blot analysis was used to measure the pAkt/Akt and pS6/S6 ratios after 1 h of incubation and p-SREBP-1, m-SREBP-1, and FASN expression after 24 h ( a ). Total Akt or S6 was used as the loading control for pAkt ( b ) or pS6 ( c ), respectively, while GAPDH was used as the loading control for p-SREBP-1 ( d ), m-SREBP-1 ( e ), and FASN ( f ).The levels are expressed as a fold value compared to control BSA-treated cells. Two-way ANOVA followed by the Bonferroni posttest was used to compare DHA and inhibitor effects. The data are presented as the mean ± S.E.M for 5 independent experiments

Article Snippet: The membranes were immunoblotted overnight at 4 °C with primary antibodies diluted in TBST; the antibodies used were rabbit monoclonal antibody against pAkt (1:1000), S6 (1:1000), or GAPDH (1:2000), rabbit polyclonal antibodies against Akt (1:1000), pS6 (1:1000), pERK1/2 (1:1000), or ERK1/2 (1:1000) (all from Cell Signaling), rabbit polyclonal anti-FASN antibodies (1:1000), or mouse monoclonal anti-SREBP1 antibody (1:200) (both from Santa Cruz).

Techniques: Expressing, Western Blot, Incubation, Control