Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular signaling pathways control mitochondrial events associated with the development of ischemia/ reperfusion-associated damage.

doi: 10.1111/j.1432-2277.2009.00883.x

Figure Lengend Snippet: Figure 1 Alterations in MAPK activity are associated with IR or HR. Representative immunoblots (a, c, e) and a summary graph (b, d) are shown. (a, b). Three animals undergoing heterotopic heart transplantation were analysed in each group. Treatment conditions were as follows: Co, untreated animals; H, 45 min of ischemia; R1-R4, hearts exposed to 45 min of ischemia and either 10 min (R1), 2 h (R2), 12 h (R3), 24 h (R4) or 48 h (R5), of reperfusion. BALB/c mouse heart lysates display low levels of ERK1,2 activity in the control group (Co). 45 min of ischemia increased stress kinase activity (p38: 4.6-fold increase, *P < 0.05 vs. control; JNK: 9.4-fold increase, *P < 0.05 vs. control) but left ERK 1,2 unaf- fected. 10 min of reperfusion (R1) caused a significant increase in ERK activity (ERK1,2: 8.4-fold increase, **P < 0.01 vs. control), and a further increase in p38 (p38: 9.4-fold increase, **P < 0.01 vs. control) and JNK (JNK1,2: 57.7-fold increase, **P < 0.01 vs. control) activity. After 2 h of reperfusion (R2) JNK (JNK1,2: 2.6-fold increase) and p38 (p38: 0.6-fold) activity had returned to control (Co) levels, while ERK (ERK1,2: 4.8-fold increase) activation still did not return to prereperfusion levels and stayed above levels in control hearts until the end of the observation period (ERK1,2: 2.73-fold increase). Data are expressed as mean ± SEM (of n = 3). (c–e) Changes in MAPK signaling in HL-1 cells and primary BALB/c car- diomyocytes subjected to 45 min of hypoxia (0.5% O2, 37 C, serum-/glucose-free medium) and up to 48 h of reoxygenation. The treatment conditions were as follows: Co, untreated cells; H, 45 min of hypoxia R1-R4: hypoxia (45 min) followed by reoxygenation in growth medium for 10 min (R1), 2 h (R2), 24 h (R3) or 48 h (R4), respectively. 45 min of hypoxia increased activity of all three MAPKs, 10 min of reperfusion lead to a further increase in their activities. After 2 h of reperfusion JNK and p38 activity had ceased while ERK activity still had not returned to control levels. Data are expressed as mean ± SEM (n = 3, all **P < 0.01). In the case of primary cardiomyocytes a single experiment was performed.

Article Snippet: Immunoblots were probed with the following antibodies: phospho-ERK (sc-7383; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho JNK (AF-1205; R&D Systems, Minneapolis, MN, USA) or phospho-p38 (9211S, Cell Signaling, Danvers, MA, USA).

Techniques: Activity Assay, Western Blot, Transplantation Assay, Control, Activation Assay

Journal: Biomedicines

Article Title: Bag-1 Protects Nucleus Pulposus Cells from Oxidative Stress by Interacting with HSP70.

doi: 10.3390/biomedicines11030863

Figure Lengend Snippet: Figure 6. Evaluation of the phosphorylation of MAPK family members and Raf-1 in NP cells. Western blotting (A) and quantification (B,C) of the phosphorylated forms of p38, ERK, JNK, and Raf-1in NP cells treated with or without H2O2 for 30 min. Data are the means ± SD n = 3, * p < 0.05.

Article Snippet: The membranes were blocked with blocking buffer (5% bovine serum albumin, 0.1% NaN3 in PBS) and incubated overnight at 4 ◦C with antibodies against Bag-1 (#ab32109; Abcam plc), p38 (#8690; Cell Signaling Technology), phosphorylated p38 (#4511; Cell Signaling Technology), ERK (#4695; Cell Signaling Technology), phosphorylated ERK (#4370; Cell Signaling Technology), JNK (#9252; Cell Signaling Technology), phosphorylated JNK (#AF1205; R&D Systems, Minneapolis, MN, USA), β-actin (#A2228; Sigma-Aldrich), or GAPDH (#G9545; Sigma-Aldrich).

Techniques: Phospho-proteomics, Western Blot

Journal: Biomedicines

Article Title: Bag-1 Protects Nucleus Pulposus Cells from Oxidative Stress by Interacting with HSP70.

doi: 10.3390/biomedicines11030863

Figure Lengend Snippet: Figure 7. Evaluation of the phosphorylation of MAPK family members and Raf-1 in Bag-1- overexpressing NP cells. Western blot analysis (A) and quantification (B,C) of the phosphorylated forms of p38, ERK, JNK, and Raf-1 in NP cells treated with or without H2O2 for 30 min. Data are the means ± SD n = 3, * p < 0.05. (D) Comparison of the expression of pERK in NP cells (NP) and Bag-1-overexpressing NP cells (Bag) without H2O2 treatment. Data are the means ± SD n = 3, * p < 0.05.

Article Snippet: The membranes were blocked with blocking buffer (5% bovine serum albumin, 0.1% NaN3 in PBS) and incubated overnight at 4 ◦C with antibodies against Bag-1 (#ab32109; Abcam plc), p38 (#8690; Cell Signaling Technology), phosphorylated p38 (#4511; Cell Signaling Technology), ERK (#4695; Cell Signaling Technology), phosphorylated ERK (#4370; Cell Signaling Technology), JNK (#9252; Cell Signaling Technology), phosphorylated JNK (#AF1205; R&D Systems, Minneapolis, MN, USA), β-actin (#A2228; Sigma-Aldrich), or GAPDH (#G9545; Sigma-Aldrich).

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

Journal: Biomedicines

Article Title: Bag-1 Protects Nucleus Pulposus Cells from Oxidative Stress by Interacting with HSP70.

doi: 10.3390/biomedicines11030863

Figure Lengend Snippet: Figure 8. Evaluation of the phosphorylation of MAPK family members and Raf-1 in thioflavin-S- treated NP cells. Western blot analysis (A) and quantification (B,C) of the phosphorylated forms of p38, ERK, JNK, and Raf-1 in thioflavin-S-treated NP cells treated with or without H2O2 for 30 min. Data are the means ± SD n = 3, * p < 0.05. (D) Comparison of the expression of pERK in NP cells (NP) and thioflavin-S-treated NP cells (ThioS) without H2O2 treatment. Data are the means ± S.D. n = 3, * p < 0.05.

Article Snippet: The membranes were blocked with blocking buffer (5% bovine serum albumin, 0.1% NaN3 in PBS) and incubated overnight at 4 ◦C with antibodies against Bag-1 (#ab32109; Abcam plc), p38 (#8690; Cell Signaling Technology), phosphorylated p38 (#4511; Cell Signaling Technology), ERK (#4695; Cell Signaling Technology), phosphorylated ERK (#4370; Cell Signaling Technology), JNK (#9252; Cell Signaling Technology), phosphorylated JNK (#AF1205; R&D Systems, Minneapolis, MN, USA), β-actin (#A2228; Sigma-Aldrich), or GAPDH (#G9545; Sigma-Aldrich).

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

Journal: Neurochemical Research

Article Title: Exogenous TIPE2 Inhibit TAK1 to Improve Inflammation and Neuropathic Pain Induced by Sciatic Nerve Injury Through Inactivating NF-κB and JNK

doi: 10.1007/s11064-022-03671-4

Figure Lengend Snippet: TIPE2 inhibited activation of NF-κB and JNK via downregulation of TAK1. A Immunofluorescence staining was performed to analyze phosphor-JNK in spinal cord and DRG after 14 days. Mean fluorescence intensity of phospho-JNK was analyzed by Image J 1.49p. The scale bar was 20 μm. B Western blot was carried out to analyze the expression of p-NF-κB p65, p-JNK1, and p-JNK2 in spinal cord after 14 days. && p < 0.01 versus Sham; % p < 0.05, %% p < 0.01 versus Model; $ p < 0.05, $$ p < 0.01 versus TIPE2-1; ! p < 0.05, !! p < 0.01 versus TIPE2-5

Article Snippet: After blocking by 5% non-fat milk, the membranes were incubated with primary antibodies against p-IκBα (1:10000, ab133462, Abcam), IκBα (1:800, ab95338, Abcam), p-JNK1(1:1200, ab47337, Abcam), p-JNK2 (1:1000, P02706, Boster), JNK1 (1:2500, ab199380, Abcam), JNK2 (1:1500, 51153-1-AP, Proteintech), β-actin (1:2000, 20536-1-AP, Proteintech), TIPE2 (1:1000, orb158628, Biorbyt), TAK1 (1:800, orb256677, Biorbyt) at 4 °C overnight.

Techniques: Activation Assay, Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing