Journal: Contrast Media & Molecular Imaging

Article Title: Lidocaine Ameliorates Diabetic Peripheral Neuropathy in Streptozotocin-Induced Diabetic Rats through Modulating the c-Jun Signaling Pathway

doi: 10.1155/2022/1888153

Figure Lengend Snippet: Primer sequences.

Article Snippet: After enclosing with 5% skimmed milk, the antibodies including anti-TNF- α (bs-10802R, 1 : 2, 000, Bioss, China), anti-IL-6 (bs-4539R, 1 : 2, 000, Bioss, China), anti-MKK4 (bs-1977R, 1 : 2, 000, Bioss, China), anti-p-MKK4 (bs-3392R, 1 : 2, 000, Bioss, China), anti-p-JNK (bsm-52462R, 1 : 2, 000, Bioss, China), anti-p-c-Jun (bs-12913R, 1 : 2, 000, Bioss, China), and anti-GAPDH (bs-0755R, 1 : 2, 000, Bioss, China) are incubated overnight.

Techniques:

Journal: Cell Death and Differentiation

Article Title: SIRT2 regulates oxidative stress-induced cell death through deacetylation of c-Jun NH 2 -terminal kinase

doi: 10.1038/s41418-018-0069-8

Figure Lengend Snippet: Deacetylation favours MKK4-dependent activation of JNK. a Graph showing the relative mRNA expression levels of JNK isoforms in wild-type (WT) and SIRT2-deficient (SIRT2-KO) mice, as measured by real-time qPCR analysis. n = 3–4 mice per group. Data are presented as mean ± s.d. *p < 0.05. Student’s t-test was used to calculate the p-values. b Graph showing the relative mRNA expression levels of JNK upstream kinases and phosphates in WT and SIRT2-KO mice, as measured by real-time qPCR analysis. n = 3–4 mice per group. Data are presented as mean ± s.d. *p < 0.05. Student’s t-test was used to calculate the p-values. c Western blotting images showing the expression of JNK upstream phosphates in HeLa cells overexpressing either control (Ad-Null) or SIRT2 (Ad-SIRT2). Similarly, the phosphorylation of MKK4, an upstream kinase of JNK, was measured by western blotting. n = 3 independent experiments. d Representative western blotting images showing MKK4 interaction with JNK. MKK4 was immunoprecipitated from cells transiently expressing SIRT2 or SIRT2-N168A mutant and probed for JNK by western blotting. Whole-cell lysates (WCLs) were probed for the overexpression of SIRT2 and endogenous JNK levels by western blotting. n = 3 independent experiments. e Representative western blotting images showing the effect of acetylation on the MKK4-mediated phosphorylation of JNK. Endogenous acetylated JNK was immunoprecipitated from HeLa cells treated with deacetylase inhibitors. The acetyl-JNK was further incubated with SIRT2 in the presence or absence of NAD+ in a test tube and then subjected to a kinase assay, where deacetylated JNK was used as substrate for MKK4. Phosphorylation and acetylation of JNK were assessed using western blotting. n = 3 independent experiments. f Representative western blotting images showing the effect of SIRT2-mediated deacetylation on the MKK4-dependent phosphorylation of JNK. MKK4 was overexpressed in HeLa cells transiently expressing either SIRT2 or SIRT2-H187Y. WCLs were probed for the overexpression of SIRT2, SIRT2-H187Y and MKK4. Endogenous JNK levels and its phosphorylation was assessed by western blotting. Lower panel shows the acetylation status of JNK assessed by western blotting

Article Snippet: Antibodies used are as follows: Akt1/2/3 (sc-8312), c-Jun (sc-1694), p-c-Jun (sc-822), GAPDH (sc-25778), JNK (sc-571), p-JNK (sc-6254) from Santacruz Biotech, DUSP1, DUSP3, DUSP6 from Cloud-Clone Corp. Ac-Lys (9681), p-Akt (Ser473; 4060), p-Akt (Thr308; 13038), c-Jun Fusion (6093), p-JNK (9251), SIRT2 (12650), β-Actin (12262) from Cell signaling, Acetyl Lysine (06-933), JNK (2470917), p300 (2328343), p-JNK (3049) from Millipore, MKK4, DUSP4 and p-MKK4 are from Thermo Fisher Scientific.

Techniques: Activation Assay, Expressing, Western Blot, Immunoprecipitation, Mutagenesis, Over Expression, Histone Deacetylase Assay, Incubation, Kinase Assay

Journal: Scientific reports

Article Title: Hyperactive TGF-β Signaling in Smooth Muscle Cells Exposed to HIV-protein(s) and Cocaine: Role in Pulmonary Vasculopathy.

doi: 10.1038/s41598-017-10438-3

Figure Lengend Snippet: Figure 4. Activation of SMAD independent TAK1 dependent MKK4-JNK axis in Tat and cocaine treated HPASMCs. (a–d) Quiescent HPASMCs were treated with cocaine (Coc) and/or Tat for 2 days followed by protein extraction and western blot. Graphs represent average densitometry of 3 independent experiments (mean ± SEM). (e) MTS cell proliferation assay was conducted on 48 h cocaine and/or Tat treated HPASMCs plated as described in Figs 1 and 2 in presence or absence of JNK inhibitor SP600125 or p38MAPK inhibitor SB203580. *p ≤ 0.05,**p ≤ 0.01,***p ≤ 0.001 compared to control, $$p ≤ 0.01 compared to Tat; ##p ≤ 0.01, ###p ≤ 0.001 compared to Coc, @p ≤ 0.001 compared to C + T.

Article Snippet: Cells were lysed with RIPA buffer (Santa Cruz Biotechnology, Dallas, TX) followed by western blot for antibodies against TGFβRI, TGFβRII, BMPR-2, total and phosphorylated (p)-SMAD2/3, TAK1 (Santa Cruz Biotechnology, Dallas, TX), p-TAK1, p-MKK3/6, p-MKK4, p-p38MAPK, p-JNK1 (Cell signaling, Beverly, MA), p-TGFβRI (Abcam, Cambridge, MA), p-TGFβRII (Biorbyt, San Francisco, CA) and β-actin (Sigma-Aldrich, St. Louis, MO).

Techniques: Activation Assay, Protein Extraction, Western Blot, Proliferation Assay, Control

Journal: Molecular Neurodegeneration

Article Title: Ubiquitin-specific protease 14 regulates c-Jun N-terminal kinase signaling at the neuromuscular junction

doi: 10.1186/1750-1326-10-3

Figure Lengend Snippet: Inhibition of USP14’s DUB activity leads to enhanced activation of the MLK3 signaling cascade. (A) Representative immunoprecipitates (IP) of MLK3 from spinal cord lysates from 4- to 6-week-old wild type and Tg Usp14CA mice, immunoblotted (IB) for K63-linked ubiquitin, phospho-serine/threonine, and MLK3. (B) Quantitation of (A), K63- and pSer/Thr- modified MLK3 was normalized to total immunoprecipitated MLK3. (C) Representative immunoblots of wild type and Tg Usp14CA spinal cord extracts probed for pMKK4 and total MKK4. β-tubulin was used as a loading control. (D) Quantitation of (C), pMKK4 was normalized to MKK4. (E) Representative immunoblots from spinal cords from 4- to 6-week-old wild type, Tg Usp14CA , ax J , and Tg Usp14 mice, probed for pJNK1/2 and total JNK1/2. JNK 1 and 2 migrate to 46 and 54 kDa, respectively. β-tubulin was used as a loading control. (F) Quantitation of (E), pJNK was normalized to JNK and quantitation includes both the 46 and 54 kDa bands. Data in (B), (D), and (F) are shown as mean ± SEM, and n = 3 animals per genotype. Symbols represent Mann-Whitney tests compared against wild type mice and corrected for multiple comparisons with a Bonferonni adjustment where appropriate; *p < 0.05, **p < 0.01, ***p < 0.001. (G) Representative images of whole-mount immunostaining of TA muscles from 4-week-old wild type and Tg Usp14CA mice using a pJNK antibody (blue). AChRs were labeled with rhodamine-conjugated α-BTX (red), and motor neuron axons were visualized via expression of YFP under the Thy1.2 promoter (green). Scale bars = 50 μm. (H) As in (G), except that scale bars = 20 μm. See also Additional file .

Article Snippet: The following antibodies were used: USP14 [ ]; β-tubulin (Developmental Studies Hybridoma Bank, Iowa City, IA); Rpt1 and MLK3 (Santa Cruz Biotechnologies, Dallas, TX); Ubiquitin (UAB Hybridoma Facility, Birmingham, AL), K48 Ubiquitin and K63 Ubiquitin (Millipore, Billerica, MA); pMKK4, MKK4, pJNK, and JNK (Cell Signaling Technology, Danvers, Massachusetts).

Techniques: Inhibition, Activity Assay, Activation Assay, Ubiquitin Proteomics, Quantitation Assay, Modification, Immunoprecipitation, Western Blot, Control, MANN-WHITNEY, Immunostaining, Muscles, Labeling, Expressing