Journal: Cell Biochemistry and Biophysics

Article Title: Ameliorating Inflammation in Insulin-resistant Rat Adipose Tissue with Abdominal Massage Regulates SIRT1/NF-κB Signaling

doi: 10.1007/s12013-022-01085-1

Figure Lengend Snippet: AM activation of the SIRT1/NF-κB pathway reduces inflammation in adipose tissue of rats subjected to HFD induced insulin resistance. Here are the protein expression levels for SIRT1 and NF-κB in adipose tissues of insulin-resistant rats induced by high-fat diets. Statistically significant differences with NFD groups were at Δ p < 0.05, and significant differences with HFD groups were at * p < 0.05. (NFD:Diet with normal fat, HFD:Fat-rich diet, RSV:resveratrol, AM:abdominal massage)

Article Snippet: In TBST (Tris-buffered saline with tween) 5% nonfat milk was applied for an hour to PVDF membranes at room temperature, and Incubation of the primary antibodies (1:500) overnight at 4 °C on a shaking plate: The sirtuin 1 (600-401-gw4), the NF-κB (100-4165) primary antibodies available from Rockland (Philadelphia, USA) and β-actin (W16197A) primary antibody (Biolegend, San Diego, CA, USA) were used as loading controls.

Techniques: Activation Assay, Expressing

Journal:

Article Title: Nuclear factor kappa ? in the dorsal raphe of macaques: an anatomical link for steroids, cytokines and serotonin

doi:

Figure Lengend Snippet: Fig. 1: Representative montages (left) from 1 level of 1 animal in each treatment group stained for NLS-NFκB with an anti-human antibody from Rockland Immunochemicals (Gilbertsville, Pa.). The antibody was made in rabbit and used at a titre of 1/50. After generation of the montage, the area was outlined and then magnified for cell counting, as shown on the right. E = estrogen, NLS-NFκB = nuclear location–specific nuclear factor kappa B, Ovx = ovariectomized, P = progesterone.

Article Snippet: The slides were then washed with phosphate-buffered saline (PBS, 0.05 mol/L phosphate, 0.14 mol/L NaCl, pH 7.3), blocked with normal goat serum and incubated with a rabbit antibody to NLS-NFκB (primary rabbit anti-human-NLS-NFκB [Rockland Immunochemicals, Gilbertsville, Pa.] at a titre of 1/50) in PBS for 48 hours at 4°C.

Techniques: Staining, Cell Counting

Journal:

Article Title: Nuclear factor kappa ? in the dorsal raphe of macaques: an anatomical link for steroids, cytokines and serotonin

doi:

Figure Lengend Snippet: Fig. 2: Top: histogram illustrating the mean (and standard error of the mean [SEM]) total number of cells that stained positive for NLS-NFκB in the DRN in each of the treatment groups. There was a significant difference between the groups (ANOVA, F3,12 = 8.16, p = 0.003). Post hoc pairwise comparisons with the Student– Newman–Keuls test indicated that the E-and E+P-treated groups had significantly fewer cells stained positive for NLS-NFκB than the placebo group. The E+P group also had significantly fewer cells stained positive for NLS-NFκB than the P-only treated group. Bottom: histogram illustrating the mean (and SEM) density of NLS-NFκB-positive cells in the DRN of each treatment group. There was a significant difference between the groups (ANOVA, F3,12 = 5.52, p = 0.013). Post hoc pairwise comparisons with the Student–Newman–Keuls test indicated that the E-and E+P-treated groups had a significantly lower density of cells stained positive for NLS-NFκB than the placebo group and the P-only treated group. ANOVA = analysis of variance, DRN = dorsal raphe nucleus, E = estrogen, NLS-NFκB = nuclear location–specific nuclear factor kappa B, Ovx = ovariectomized, P = progesterone. a,d,f = p < 0.05, b,c,e,g = p < 0.01.

Article Snippet: The slides were then washed with phosphate-buffered saline (PBS, 0.05 mol/L phosphate, 0.14 mol/L NaCl, pH 7.3), blocked with normal goat serum and incubated with a rabbit antibody to NLS-NFκB (primary rabbit anti-human-NLS-NFκB [Rockland Immunochemicals, Gilbertsville, Pa.] at a titre of 1/50) in PBS for 48 hours at 4°C.

Techniques: Staining

Journal:

Article Title: Nuclear factor kappa ? in the dorsal raphe of macaques: an anatomical link for steroids, cytokines and serotonin

doi:

Figure Lengend Snippet: Fig. 3: Photomicrographs illustrating NLS-NFκB immunostaining in the DRN after antigen retrieval versus no antigen retrieval. Left panels: preincubation of sections with antigen-unmasking buffer and microwave treatment for 6 min yields widespread cytoplasmic staining with intense nucleolus staining even at a dilute titre of antibody (1/200). Right panels: without antigen retrieval, there is sparser immunostaining that is largely nuclear or perinuclear even at a concentrated titre of antibody (1/50). The antigen-retrieval buffer apparently reveals the large amount of NFκB present in the cell, which is normally bound to IκB. Omission of the primary antibody or further dilution of the primary antibody eliminated staining. DRN = dorsal raphe nucleus, NLS-NFκB = nuclear location–specific nuclear factor kappa B.

Article Snippet: The slides were then washed with phosphate-buffered saline (PBS, 0.05 mol/L phosphate, 0.14 mol/L NaCl, pH 7.3), blocked with normal goat serum and incubated with a rabbit antibody to NLS-NFκB (primary rabbit anti-human-NLS-NFκB [Rockland Immunochemicals, Gilbertsville, Pa.] at a titre of 1/50) in PBS for 48 hours at 4°C.

Techniques: Immunostaining, Staining

Journal:

Article Title: Nuclear factor kappa ? in the dorsal raphe of macaques: an anatomical link for steroids, cytokines and serotonin

doi:

Figure Lengend Snippet: Fig. 4: Panels A–D illustrate cells in the DRN (A,B,C) and descending tract or medial longitidunal fasciculus (D) stained for NFκB p65 with an antibody raised in goat from Santa Cruz Biotechnology (Santa Cruz, Calif.). The nuclei of cells that are positive are highlighted with a black arrow, and negative cells are highlighted with an orange arrow. The picture in panel A is magnified 200-fold, and the pictures in panels B–D are magnified 400-fold. Panels E–G illustrate cells from the DRN that were double-labelled for NFκB p65 (nuclei) and serotonin (cytoplasm). The nuclei are stained black/dark brown and the cytoplasm is stained light brown in positive cells. The double-headed black arrows indicate double-labelled cells. The single-headed black arrow indicates a cell that only contains NFκB p65–positive nuclei. The green arrow indicates a cell that appears to be positive for serotonin but lacks a nucleus. The orange arrow indicates a cell that is negative for both NFκB p65 and serotonin. The pictures in panels E–G are magnified 600-fold. DRN = dorsal raphe nucleus, NFκB = nuclear location–specific nuclear factor kappa B.

Article Snippet: The slides were then washed with phosphate-buffered saline (PBS, 0.05 mol/L phosphate, 0.14 mol/L NaCl, pH 7.3), blocked with normal goat serum and incubated with a rabbit antibody to NLS-NFκB (primary rabbit anti-human-NLS-NFκB [Rockland Immunochemicals, Gilbertsville, Pa.] at a titre of 1/50) in PBS for 48 hours at 4°C.

Techniques: Staining

Journal:

Article Title: Nuclear factor kappa ? in the dorsal raphe of macaques: an anatomical link for steroids, cytokines and serotonin

doi:

Figure Lengend Snippet: Fig. 5: Panel A: linearity of NFκB p65 signal is reflected by increased signal density with increasing amounts of protein loaded. The protein is an extract of RN46 cells. Similar results were obtained with 50, 100, 200 and 300 (not shown) μg of protein from the dorsal raphe region. Panel B: a representative film from a Western blot for NFκB p65 on extracts of monkey raphe region is shown. One animal from each treatment group was included, as well as an extra ovariectomized control animal and an extract of RN46 cells. Each monkey sample contained 200 μg of protein, and the RN46 cell extract contained 100 μg of protein. The same arrangement was repeated on 4 blots to assay the 4 animals in each treatment group. Panel C: Histogram illustrating the average optical density (and standard error of the mean [SEM]) of all of the treatment groups. There was no difference between the groups (analysis of variance, p > 0.1). E = estrogen, NLS-NFκB = nuclear location–specific nuclear factor kappa B, OD = optical density, Ovx = ovariectomized, P = progesterone, Ral = raloxifene.

Article Snippet: The slides were then washed with phosphate-buffered saline (PBS, 0.05 mol/L phosphate, 0.14 mol/L NaCl, pH 7.3), blocked with normal goat serum and incubated with a rabbit antibody to NLS-NFκB (primary rabbit anti-human-NLS-NFκB [Rockland Immunochemicals, Gilbertsville, Pa.] at a titre of 1/50) in PBS for 48 hours at 4°C.

Techniques: Western Blot, Control

Journal:

Article Title: Nuclear factor kappa ? in the dorsal raphe of macaques: an anatomical link for steroids, cytokines and serotonin

doi:

Figure Lengend Snippet: Fig. 6: Histogram illustrating the relative abundance (and standard error of the mean [SEM]) of NFκB p65 in spayed macaques treated with placebo (ovx), estrogen for 28 days (E) or estrogen for 28 days supplemented with progesterone for the last 14 days (E+P). There was no difference in the ratio of NFκB:GAPDH between the groups (analysis of variance, p > 0.1). GAPDH = glyceraldehyde -3-phosphate dehydrogenase gene, NLS-NFκB = nuclear location –specific nuclear factor kappa B, Ovx = ovariectomized.

Article Snippet: The slides were then washed with phosphate-buffered saline (PBS, 0.05 mol/L phosphate, 0.14 mol/L NaCl, pH 7.3), blocked with normal goat serum and incubated with a rabbit antibody to NLS-NFκB (primary rabbit anti-human-NLS-NFκB [Rockland Immunochemicals, Gilbertsville, Pa.] at a titre of 1/50) in PBS for 48 hours at 4°C.

Techniques: