total cpla2  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: Modified lipid metabolism and cytosolic phospholipase A2 activation in mesangial cells under pro-inflammatory conditions

doi: 10.1038/s41598-022-10907-4

Figure Lengend Snippet: Activation of rate-limiting enzyme cPLA2, upstream of COX-2. Phospholipase cPLA2 is upregulated and phosphorylated in human mesangial cells after 24 h stimulations with IL-1β and PDGF-BB ( a ). cPLA2 activity is accordingly increased ( b ). The activity in the controls was below the LOD of 3.5 nmol/min/ml. In this case, the LOD value was plotted in the graph. PLA2G4A gene expression was already upregulated after 4 h in both treatments ( c , d ). * P < 0.05; *** P < 0.001. Data are reported as mean ± SEM. Uncropped blots after chemiluminescence development and total protein stain free blots are presented in Supplementary Fig. d. Quantification of the bands in the western blot is reported in Supplementary Fig. .

Article Snippet: The following antibodies were used: anti-Phospho-cPLA2 #53044 (Ser505) and anti-total cPLA2 #2831 (Cell Signaling technology, Danvers, MA), used at a 1:500 v/v dilution in TBST 5% BSA; anti-Sphk1 #12071, anti-NLRP3 #15101, anti-IL-1β #12703, anti-COX-2 #4842 (Cell Signaling technology) used at a 1:500 v/v dilution in 5% milk; finally, anti-Cerk #HPA064699 (Sigma Aldrich), used at a 1:1000 v/v dilution in TBST 5% milk.

Techniques: Activation Assay, Activity Assay, Gene Expression, Staining, Western Blot

Journal: Scientific Reports

Article Title: Modified lipid metabolism and cytosolic phospholipase A2 activation in mesangial cells under pro-inflammatory conditions

doi: 10.1038/s41598-022-10907-4

Figure Lengend Snippet: Inhibition of cPLA2 with AACOCF3 1 μM reduces proliferation, secretion, and prostaglandin secretion. AACOCF3 reduced PDGF-BB driven cell proliferation ( a ) and migration ( b ). No effect was observed when PGE2 and iloprost (PGI2) were administered together (mimicking their secretion levels obtained after 24 h IL-1β stimulation of mesangial cells) and at low PGE2 concentrations (mimicking PGE2 secretion levels after 24 h PDGF-BB stimulation) ( b ). Prostaglandin administration had no migratory effect on mesangial cells ( b ). * P < 0.05; ** P < 0.01; *** P < 0.001, rlu relative light units. Data are reported as mean ± SEM.

Article Snippet: The following antibodies were used: anti-Phospho-cPLA2 #53044 (Ser505) and anti-total cPLA2 #2831 (Cell Signaling technology, Danvers, MA), used at a 1:500 v/v dilution in TBST 5% BSA; anti-Sphk1 #12071, anti-NLRP3 #15101, anti-IL-1β #12703, anti-COX-2 #4842 (Cell Signaling technology) used at a 1:500 v/v dilution in 5% milk; finally, anti-Cerk #HPA064699 (Sigma Aldrich), used at a 1:1000 v/v dilution in TBST 5% milk.

Techniques: Inhibition, Migration

Journal: Scientific Reports

Article Title: Modified lipid metabolism and cytosolic phospholipase A2 activation in mesangial cells under pro-inflammatory conditions

doi: 10.1038/s41598-022-10907-4

Figure Lengend Snippet: Inhibition of cPLA2 with AACOCF3 1 μM reduces IL-1β, PDGF-BB induced PGE2 secretion. ** P < 0.01. Data are reported as mean ± SEM.

Article Snippet: The following antibodies were used: anti-Phospho-cPLA2 #53044 (Ser505) and anti-total cPLA2 #2831 (Cell Signaling technology, Danvers, MA), used at a 1:500 v/v dilution in TBST 5% BSA; anti-Sphk1 #12071, anti-NLRP3 #15101, anti-IL-1β #12703, anti-COX-2 #4842 (Cell Signaling technology) used at a 1:500 v/v dilution in 5% milk; finally, anti-Cerk #HPA064699 (Sigma Aldrich), used at a 1:1000 v/v dilution in TBST 5% milk.

Techniques: Inhibition

Journal: Scientific Reports

Article Title: Modified lipid metabolism and cytosolic phospholipase A2 activation in mesangial cells under pro-inflammatory conditions

doi: 10.1038/s41598-022-10907-4

Figure Lengend Snippet: Signaling pathway overview. Hyperglycemia stimulates mesangial production of IL-1β which activates PDGF-BB secretion. In turn, PDGF-BB promotes IL-1β secretion, sustaining and boosting the pathway activation. IL-1β and PDGF-BB stimulation give rise to the production of phosphorylated sphingoid bases, activating COX-2 transcription. At the same time, IL-1β and PDGF-BB stimulate cPLA2 activation. Arachidonic acid (AA) released by cPLA2 is converted into prostanoids by COX-2 and downstream enzymes. The other product of cPLA2, lysophosphatidylcholine (LPC), is converted into lysophosphatidic acid (LPA) by autotaxin. Thus, hyperglycemia triggered the activation of an interplay between IL-1β and PDGF-BB which stimulates the secretion of lipid hormones (prostanoids PGE2, PGI2, but also lysophosphatidic acid) with hemodynamic, proliferative, and migratory effects at glomerular level. Inhibition of the cPLA2 reaction with AACOCF3 blocks the supply of AA for the COX-2 reaction, thus resolving the inflammatory stimulus. In addition, LPC is not produced, and this blocks the supply for the autotaxin reaction and LPA mediated proliferative response.

Article Snippet: The following antibodies were used: anti-Phospho-cPLA2 #53044 (Ser505) and anti-total cPLA2 #2831 (Cell Signaling technology, Danvers, MA), used at a 1:500 v/v dilution in TBST 5% BSA; anti-Sphk1 #12071, anti-NLRP3 #15101, anti-IL-1β #12703, anti-COX-2 #4842 (Cell Signaling technology) used at a 1:500 v/v dilution in 5% milk; finally, anti-Cerk #HPA064699 (Sigma Aldrich), used at a 1:1000 v/v dilution in TBST 5% milk.

Techniques: Activation Assay, Inhibition, Produced

Journal: Journal of Neuroinflammation

Article Title: WWL70 protects against chronic constriction injury-induced neuropathic pain in mice by cannabinoid receptor-independent mechanisms

doi: 10.1186/s12974-017-1045-9

Figure Lengend Snippet: WWL70 treatment had no effect on cPLA 2 phosphorylation in CCI mouse sciatic nerve. a Representative blot of two tissue lysates made from sciatic nerve removed from sham and CCI injured mice at 7 days after injury probed with antisera specifically recognizing p-cPLA 2 or total cPLA 2 . b Quantification of western blot data showing the relative ratio of p-cPLA 2 /cPLA 2 each normalized to β-actin (mean ± S.E.M., n = 4)

Article Snippet: Thereafter, proteins were transferred onto nitrocellulose membranes, blocked for 1 h at RT with 5% non-fat milk, and incubated overnight at 4 °C with the primary rabbit antibodies against phosphorylated cPLA 2 (1:300; Cat# 2831, Cell Signaling) and total cPLA 2 (1:1000; Cat# 2832, Cell Signaling).

Techniques: Western Blot