phosphorylated 46 54 jnk  (Cell Signaling Technology Inc)

Journal: Neurobiology of aging

Article Title: APOE4 homozygote females are resistant to the beneficial effects of 17β-estradiol on memory and CA1 dendritic spine density in the EFAD mouse model of Alzheimer’s disease

doi: 10.1016/j.neurobiolaging.2022.06.005

Figure Lengend Snippet: Levels of phosphorylated ERK and JNK were unaffected by E 2 treatment or genotype

Article Snippet: Membranes were blocked in 5% milk and incubated with following primary antibodies overnight at 4°C: phosphorylated 42/44 ERK (#9101, 1:2,000, Cell Signaling Technology), total ERK (#9102, 1:20,000, Cell Signaling Technology), phosphorylated 46/54 JNK (#4668, 1:1000, Cell Signaling Technology), total JNK (#9252, 1:1000, Cell Signaling Technology), ERα (H-184, 1:1000, Santa Cruz Biotechnology), ERβ (#PA1-310B, 0.25μg/mL, Thermo Fisher), and GPER (ab39742, 1:250, Abcam).

Techniques:

Journal: Cells

Article Title: Arachidonic Acid Cascade and Eicosanoid Production Are Elevated While LTC4 Synthase Modulates the Lipidomics Profile in the Brain of the HIVgp120-Transgenic Mouse Model of NeuroHIV

doi: 10.3390/cells11132123

Figure Lengend Snippet: MAPK signaling pathway in female LTC4SKO mice shows the reduced activity levels of p38 and ERK in protein brain lysates. The quantification of Western blot densitometries of: phosphorylated p38 MAPK (pp38)/total p38/tubulin (p38) ( A ); phosphorylated ERK1/2 (pERK)/total ERK1/2/tubulin (ERK) ( B ); and phosphorylated JNK1/2 (pJNK)/total JNK1/2/tubulin (JNK) ( C ) all normalized to their respective wild type control. Representative images of the blots are shown with male and female samples separated by a protein ladder lane are for p38 ( D ), ERK1/2 ( E ), and JNK1/2 ( F ). Male ( blue ) and female ( red ) data sets are shown, separated by a dashed line. Values in graphs are mean ± s.e.m.; n = 4–6 per genotype for both male and female data sets, 12 months of age. Individual data points are represented in the graphs by an ⚪ symbol. ANOVA and Fisher’s PLSD post hoc test for males and females were analyzed independently while normalized to their respective WT controls; p -value level of significance is as follows: * p ≤ 0.05, @@ p ≤ 0.01, @@@ p ≤ 0.001—while the p -value significance correlations are symbolized as follows: * indicates significances from the WT, @ indicates significances from HIVgp120tg.

Article Snippet: Antibodies phospho-p38 (1°Ab- 1:1000; anti-rabbit 2°Ab- 1:5000, 43 kDa) (Cell Signaling, Danvers, MA, USA; 9211), total p38 (1°Ab- 1:2000; anti-rabbit 2°Ab- 1:25,000, 40 kDa) (Cell Signaling; 9212), phospho-ERK1 (1°Ab- 1:1000; anti-rabbit 2°Ab- 1:5000, 42/44 kDa) (Cell Signaling; 9101), total ERK1 (1°Ab- 1:2000; anti-rabbit 2°Ab- 1:5000, 42/44 kDa) (Cell Signaling; 9102), active JNK (1°Ab- 1:1000; anti-rabbit 2°Ab- 1:3000, 46/54 kDa) (Promega, Madison, WI, USA; V793A), total JNK (1°Ab- 1:1000; anti-rabbit 2°Ab- 1:3000, 46/54 kDa) (Cell Signaling; 9252), and α-tubulin (1°Ab- 1:2000; anti-mouse 2°Ab- 1:10,000, 50 kDa) (Sigma-Aldrich, Burlington, MA, USA; T9026).

Techniques: Activity Assay, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Defining the Role of Mitochondrial Fission in Corneal Myofibroblast Differentiation

doi: 10.1167/iovs.63.4.2

Figure Lengend Snippet: Intracellular mediators underlying anti-fibrotic effects of Mdivi-1. ( A ) Representative Western blot depicting phospho- and total-SMAD3 expression levels in protein extracts from cultured cat corneal fibroblasts. Cells were incubated ± 10 µM Mdivi-1, ± 1 ng/mL TGF-β1 and finally 2.3 µM SB431542 with 1 ng/mL TGF-β1, as indicated. ( B ) Plot of densitometric ratios for phospho-SMAD3 expression relative to total-SMAD2/3, normalized to the TGF-β1 treatment. One-way repeated measures ANOVA: F(3,11) = 116.3, P < 0.0001. Significances from post-hoc Tukey's HSD tests relative to TGF-β1 alone are denoted: * P < 0.05, # P < 0.01. ( C ) Representative Western blots of corneal fibroblast extracts for phosphor-p54 and -p46 and total-JNK expression under the conditions indicated. ( D ) Plot of densitometric ratios for p-p54 JNK expression relative to total-p54 JNK, normalized to the TGF-β1 treatment. One-way repeated measures ANOVA: F(3,11) = 7.48, P = 0.01884. Significances relative to the TGF-β1 condition were computed and are denoted as in B . ( E ) Representative Western blot of corneal fibroblast extracts showing phospho-p38, phospho-ERK, and phospho-AKT expression levels under the conditions indicated. ( F ) Plot of densitometry ratios for phosphorylated proteins relative to β-actin, normalized to the TGF-β1 treatment. One-way repeated measures ANOVAs demonstrated significant effects of treatments for all 3 molecules: p38 F(3,11) = 37.52, P = 0.0003; pERK F(3,11) = 11.12, P = 0.0073; pAKT F(3,11) = 8.85, P = 0.0127. Significances from post-hoc Tukey's HSD tests relative to TGF-β1 are indicated as in B and D . For all graphs, data shown are means ± SD from 3 experimental replicates.

Article Snippet: Blots were incubated overnight at 4°C containing primary antibodies to the following targets at the dilutions indicated: Type 1 collagen (COL1; 1:2000; no. LF-68, kindly provided by Dr. Larry W. Fisher, NIH, Bethesda, MD, USA), total Fibronectin (t-FN; 1:2000; #H-300; Santa Cruz Inc.), α-SMA (1:10,000; no. MA5-11547; Thermo Fisher Scientific, Waltham, MA, USA), total SMAD 2/3 (1:2000; no. 8685; Cell Signaling Technology, Danvers, MA, USA), phosphorylated small mothers against decapentaplegic 3 (SMAD3) (S423+S425; 1:1000; no. P00059-1; Boster Technology, Pleasanton, CA, USA), phospho-p46/54 JNK Thr183/Tyr185 (1:1000; no. 4671; Cell Signaling Technology), phospho-p38 MAPK Thr180/Tyr182 (1:1000; no. 9215; Cell Signaling Technology), phospho-p44/42 MAPK Thr202/Tyr204 (1:1000; no. 9106; Cell Signaling Technology), phospho-AKT Ser473 (1:1000; no. 4060; Cell Signaling Technology), total-p46/54 JNK (1:1000; no. 9252; Cell Signaling Technology), p-Drp1 Ser616 (1:500; no. 3455; Cell Signaling Technology), total-Drp1 (1:1000; no. 611112; BD Biosciences, San Jose, CA, USA), Opa1 (1:1000; no. 612606; BD Biosciences), Mfn1 (1:1000; ab57602; Abcam, Boston, MA, USA), Mfn2 (1:1000; no. M6319; Sigma Aldrich), and β-actin-HRP (1:5000; no. sc-47778; Santa Cruz Inc.).

Techniques: Western Blot, Expressing, Cell Culture, Incubation