Journal: Alcoholism, clinical and experimental research

Article Title: Regulation of Kisspeptin Synthesis and Release in the Preoptic/Anterior Hypothalamic Region of Prepubertal Female Rats: Actions of IGF-1 and Alcohol

doi: 10.1111/acer.13539

Figure Lengend Snippet: Effects of IGF-1 and acute ALC exposure on Kp protein in the POA/RHA of prepubertal female rats. A) Representative Western blot of Kp and β- actin proteins from control (lanes 1-3), IGF-1 (lanes 4-6), ALC (7-9) and IGF-1 + ALC (lanes 10-12) animals. B) Densitometric quantification of all bands assessing the Kp protein. These data were normalized to the internal control β-actin protein. IGF-1 (checked bar) induced an increase in Kp over control (solid bar) animals. Note that 3g/kg exposure to ALC (open bar) did not alter basal expression of Kp protein levels, but the IGF-1 induced expression of Kp protein was blocked by acute ALC exposure (hatched bar). Each bar represents the mean ± SEM of the Kp/β-actin ratio. The number of animals represented by each bar is 6. **p<0.01.

Article Snippet: Following transfer, membranes were blocked with 5% nonfat dried milk/0.1% Tween 20 in PBS (pH 7.4) for 3 hr and subsequently incubated at 4 C overnight with rabbit anti-total-IRS-1 (1:500; Cohesion Biosciences Ltd, London, UK) or anti-p-IRS-1 (Ser307; 1:500; Assay Biotechnology Company, Inc, Sunnyvale, CA), rabbit anti-total (1:1000) or anti-p-Akt (Ser473, 1:3000; Cell Signaling Tech., Danvers, MA), rabbit anti-total (1:2000) or anti-p-TSC2 (Thr1462, 1:250; Cell Signaling Tech., Danvers, MA), rabbit anti-Rheb (1μg/ml; Abcam Inc., Cambridge, MA), rabbit anti-total or anti-p-mTOR (Ser2448, 1:1000; Cell Signaling Tech., Danvers, MA) and rabbit anti-Kisspeptin (3μg/ml; Novus Biologicals, Littleton, CO).

Techniques: Western Blot, Control, Expressing

Journal: Alcoholism, clinical and experimental research

Article Title: Regulation of Kisspeptin Synthesis and Release in the Preoptic/Anterior Hypothalamic Region of Prepubertal Female Rats: Actions of IGF-1 and Alcohol

doi: 10.1111/acer.13539

Figure Lengend Snippet: Effects of IGF-1 and acute ALC exposure on Akt protein in the POA/RHA of prepubertal female rats. A) Representative Western blot of phosphorylated (p)-Akt and total Akt from control (lanes 1-3), IGF-1 (lanes 4-6), ALC (7-9) and IGF-1 + ALC (lanes 10-12) animals. B) Each bar represents the mean ± SEM of the densitometric quantification of all bands assessing the p-Akt normalized to total Akt protein. IGF-1 (checked bar) stimulated an increase in p-Akt (p <0.05) over control (solid bar) animals. Note that 3g/kg exposure to ALC (open bar) did not affect phosphorylation of Akt but the IGF-1 induced expression of p-Akt was blocked by acute ALC exposure (hatched bar). The number of animals represented by each bar is 6. *p<0.05.

Article Snippet: Following transfer, membranes were blocked with 5% nonfat dried milk/0.1% Tween 20 in PBS (pH 7.4) for 3 hr and subsequently incubated at 4 C overnight with rabbit anti-total-IRS-1 (1:500; Cohesion Biosciences Ltd, London, UK) or anti-p-IRS-1 (Ser307; 1:500; Assay Biotechnology Company, Inc, Sunnyvale, CA), rabbit anti-total (1:1000) or anti-p-Akt (Ser473, 1:3000; Cell Signaling Tech., Danvers, MA), rabbit anti-total (1:2000) or anti-p-TSC2 (Thr1462, 1:250; Cell Signaling Tech., Danvers, MA), rabbit anti-Rheb (1μg/ml; Abcam Inc., Cambridge, MA), rabbit anti-total or anti-p-mTOR (Ser2448, 1:1000; Cell Signaling Tech., Danvers, MA) and rabbit anti-Kisspeptin (3μg/ml; Novus Biologicals, Littleton, CO).

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

Journal: Alcoholism, clinical and experimental research

Article Title: Regulation of Kisspeptin Synthesis and Release in the Preoptic/Anterior Hypothalamic Region of Prepubertal Female Rats: Actions of IGF-1 and Alcohol

doi: 10.1111/acer.13539

Figure Lengend Snippet: Effects of IGF-1 and acute ALC exposure on TSC2 and Rheb proteins in the POA/RHA of prepubertal female rats. A) Representative Western blot of phosphorylated (p)- and total-TSC2 proteins from control (lanes 1-3), IGF-1 (lanes 4-6), ALC (7-9) and IGF-1 + ALC (lanes 10-12) animals. B) Each bar represents the mean ± SEM of the densitometric quantification of all bands assessing p-TSC2 protein normalized to total TSC2 protein. C) Representative Western blot of Rbeb and β-actin proteins from control (lanes 1-3), IGF-1 (lanes 4-6), ALC (7-9) and IGF-1 + ALC (lanes 10-12) animals. D) Densitometric quantification of all bands assessing Rbeb protein normalized to β-actin protein. IGF-1 (checked bars) induced an increase in p-TSC2 and Rheb over control (solid bars) animals. Note that 3g/kg exposure to ALC (open bars) did not alter basal expression of either protein, but the IGF-1-induced increase of p-TSC2 and Rheb proteins were both blocked by acute ALC exposure (hatched bars).The number of animals represented by each bar is 6. **p<0.01, *p<0.05.

Article Snippet: Following transfer, membranes were blocked with 5% nonfat dried milk/0.1% Tween 20 in PBS (pH 7.4) for 3 hr and subsequently incubated at 4 C overnight with rabbit anti-total-IRS-1 (1:500; Cohesion Biosciences Ltd, London, UK) or anti-p-IRS-1 (Ser307; 1:500; Assay Biotechnology Company, Inc, Sunnyvale, CA), rabbit anti-total (1:1000) or anti-p-Akt (Ser473, 1:3000; Cell Signaling Tech., Danvers, MA), rabbit anti-total (1:2000) or anti-p-TSC2 (Thr1462, 1:250; Cell Signaling Tech., Danvers, MA), rabbit anti-Rheb (1μg/ml; Abcam Inc., Cambridge, MA), rabbit anti-total or anti-p-mTOR (Ser2448, 1:1000; Cell Signaling Tech., Danvers, MA) and rabbit anti-Kisspeptin (3μg/ml; Novus Biologicals, Littleton, CO).

Techniques: Western Blot, Control, Expressing

Journal: Alcoholism, clinical and experimental research

Article Title: Regulation of Kisspeptin Synthesis and Release in the Preoptic/Anterior Hypothalamic Region of Prepubertal Female Rats: Actions of IGF-1 and Alcohol

doi: 10.1111/acer.13539

Figure Lengend Snippet: Effects of IGF-1 and acute ALC exposure on mTOR protein in the POA/RHA of prepubertal female rats. A) Representative Western blot of phosphorylated (p)-mTOR and total mTOR from control (lanes 1-3), IGF-1 (lanes 4-6), ALC (7-9) and IGF-1 + ALC (lanes 10-12) animals. B) Each bar represents the mean ± SEM of the densitometric quantification of all bands assessing the p-mTOR normalized to total mTOR protein. IGF-1 (checked bar) stimulated an increase in p-mTOR over control (solid bar) animals. Note that 3g/kg exposure to ALC (open bar) did not affect phosphorylation of mTOR but the IGF-1 induced expression of p-mTOR was blocked by acute ALC exposure (hatched bar). The number of animals represented by each bar is 6. *p<0.05.

Article Snippet: Following transfer, membranes were blocked with 5% nonfat dried milk/0.1% Tween 20 in PBS (pH 7.4) for 3 hr and subsequently incubated at 4 C overnight with rabbit anti-total-IRS-1 (1:500; Cohesion Biosciences Ltd, London, UK) or anti-p-IRS-1 (Ser307; 1:500; Assay Biotechnology Company, Inc, Sunnyvale, CA), rabbit anti-total (1:1000) or anti-p-Akt (Ser473, 1:3000; Cell Signaling Tech., Danvers, MA), rabbit anti-total (1:2000) or anti-p-TSC2 (Thr1462, 1:250; Cell Signaling Tech., Danvers, MA), rabbit anti-Rheb (1μg/ml; Abcam Inc., Cambridge, MA), rabbit anti-total or anti-p-mTOR (Ser2448, 1:1000; Cell Signaling Tech., Danvers, MA) and rabbit anti-Kisspeptin (3μg/ml; Novus Biologicals, Littleton, CO).

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

Journal: Alcoholism, clinical and experimental research

Article Title: Regulation of Kisspeptin Synthesis and Release in the Preoptic/Anterior Hypothalamic Region of Prepubertal Female Rats: Actions of IGF-1 and Alcohol

doi: 10.1111/acer.13539

Figure Lengend Snippet: Effects of IGF-1 and acute ALC exposure on IRS-1 protein in the POA/RHA of prepubertal female rats. A) Representative Western blot of phosphorylated (p)- and total-IRS-1 proteins from control (lanes 1-3), IGF-1 (lanes 4-6), ALC (7-9) and IGF-1 + ALC (lanes 10-12) animals. B) Each bar represents the mean ± SEM of the densitometric quantification of all bands assessing p-IRS-1 protein normalized to total IRS-1 protein. IGF-1 (checked bar) induced an increase in p-IRS-1 over control (solid bar) animals. Note that 3g/kg exposure to ALC (open bar) did not alter basal expression of p-IRS-1, but that the IGF-1 induced increase of p-IRS-1was blocked by acute ALC exposure (hatched bar). The number of animals represented by each bar is 6. **p<0.01.

Article Snippet: Following transfer, membranes were blocked with 5% nonfat dried milk/0.1% Tween 20 in PBS (pH 7.4) for 3 hr and subsequently incubated at 4 C overnight with rabbit anti-total-IRS-1 (1:500; Cohesion Biosciences Ltd, London, UK) or anti-p-IRS-1 (Ser307; 1:500; Assay Biotechnology Company, Inc, Sunnyvale, CA), rabbit anti-total (1:1000) or anti-p-Akt (Ser473, 1:3000; Cell Signaling Tech., Danvers, MA), rabbit anti-total (1:2000) or anti-p-TSC2 (Thr1462, 1:250; Cell Signaling Tech., Danvers, MA), rabbit anti-Rheb (1μg/ml; Abcam Inc., Cambridge, MA), rabbit anti-total or anti-p-mTOR (Ser2448, 1:1000; Cell Signaling Tech., Danvers, MA) and rabbit anti-Kisspeptin (3μg/ml; Novus Biologicals, Littleton, CO).

Techniques: Western Blot, Control, Expressing

Journal: mBio

Article Title: The Retropepsin-Type Protease APRc as a Novel Ig-Binding Protein and Moonlighting Immune Evasion Factor of Rickettsia

doi: 10.1128/mBio.03059-21

Figure Lengend Snippet: APRc protects E. coli from complement-mediated killing, and interaction with IgG contributes to serum resistance. (A) Western blot analysis with anti-APRc antibody of total protein extracts from BL21 Star(DE3) E. coli strain expressing the empty vector backbone pET28a (pET) or the plasmid encoding untagged full-length APRc wild type (APRcFL) and the corresponding catalytic mutant (APRcFL_D140N). (B) The serum-sensitive BL21 Star(DE3) E. coli strain expressing the empty vector (pET) or the plasmid encoding APRcFL and APRcFL_D140N was independently incubated 1:1 with PBS or in PBS containing 40% NHS for 1 h. After incubation, the samples were serially diluted, plated onto LB agar plates, and incubated overnight at 37°C. The average number of CFU per milliliter was calculated from the replicate plate counts. The data are presented as survival rate, which was calculated as the percentage of the original cell number at T 0 (considered 100% survival). Data represent the mean ± SD from 4 independent biological replicates. Significance was determined using a one-way ANOVA followed by Tukey multiple-comparison test using GraphPad Prism 8 (***, P < 0.001; ****, P < 0.0001). (C) BL21 Star(DE3) E. coli strain expressing the empty vector (pET) or the plasmid encoding APRcFL_D140N was independently incubated 1:1 with PBS or in PBS containing 40% NHS or 40% NHSΔIgG/IgM for 1 h. After incubation, samples were treated as described for panel B. Data represent the mean ± SD from 4 independent biological replicates. (D) Flow cytometry analysis to query for human IgG deposition at the surface of E. coli cells. PFA-fixed E. coli expressing the untagged full-length APRc catalytic mutant (APRcFL_D140N) was incubated with HBSS (gray, dotted trace), HBSS containing 40% NHS (blue trace), and HBSS containing 40% NHSΔIgG/IgM (pink trace) for 1 h, followed by detection using anti-human IgG (Fc-specific)-FITC antibody. (E and F) Flow cytometry analysis querying for complement C3 (E) and IgG (F) deposition at the surface of R. massiliae . PFA-fixed R. massiliae was incubated with HBSS (gray, dotted trace), HBSS containing 50% NHS (blue trace), and HBSS containing 50% NHSΔIgG/IgM (pink trace) for 1 h, followed by detection using anti-complement C3 and secondary detection with goat anti-rabbit FITC-conjugated antibody or anti-human IgG (Fc-specific)-FITC antibody, respectively.

Article Snippet: Upon expression, bacteria were resuspended in PBS to an OD 600 of 0.2, and 100 μl of this suspension was diluted 1:1 with PBS, 40% NHS, 40% human IgG/IgM-depleted serum (NHSΔIgG/IgM) (34010-10; Pel-Freez Biologicals LLC), followed by incubation for 1 h at 37°C with rotation.

Techniques: Western Blot, Expressing, Plasmid Preparation, Mutagenesis, Incubation, Flow Cytometry